There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a ...vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1-4 (RXFP1-4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.
While being an important component of normal cellular function, excess levels of reactive oxygen species (ROS) cause cell damage and death. The ability to protect sperm against oxidative damage is of ...particular importance in the artificial reproduction industry because of the increased production of ROS by the sperm cell during processing. This review discusses the formation of ROS and the use of antioxidants in protecting boar sperm against oxidative damage.
Relaxin-3 is a neuropeptide that is abundantly expressed by discrete brainstem neuron populations that broadly innervate forebrain areas rich in the relaxin-3 G-protein-coupled-receptor, RXFP3. Acute ...and subchronic central administration of synthetic relaxin-3 or an RXFP3-selective agonist peptide, R3/I5, increase feeding and body weight in rats. Intrahypothalamic injection of relaxin-3 also increases feeding. In this study, we developed a recombinant adeno-associated virus 1/2 (rAAV1/2) vector that drives expression and constitutive secretion of bioactive R3/I5 and assessed the effect of intrahypothalamic injections on daily food intake and body weight gain in adult male rats over 8 weeks. In vitro testing revealed that the vector rAAV1/2-fibronectin (FIB)-R3/I5 directs the constitutive secretion of bioactive R3/I5 peptide. Bilateral injection of rAAV1/2-FIB-R3/I5 vector into the paraventricular nucleus produced an increase in daily food intake and body weight gain (P<0.01, ~23%, respectively), relative to control treatment. In a separate cohort of rats, quantitative polymerase chain reaction analysis of hypothalamic mRNA revealed strong expression of R3/I5 transgene at 3 months post-rAAV1/2-FIB-R3/I5 infusion. Levels of mRNA transcripts for the relaxin-3 receptor RXFP3, the hypothalamic 'feeding' peptides neuropeptide Y, AgRP and POMC, and the reproductive hormone, GnRH, were all similar to control, whereas vasopressin and oxytocin (OT) mRNA levels were reduced by ~25% (P=0.051) and ~50% (P<0.005), respectively, in rAAV1/2-FIB-R3/I5-treated rats (at 12 weeks, n=9/8 rats per group). These data demonstrate for the first time that R3/I5 is effective in modulating feeding in the rat by chronic hypothalamic RXFP3 activation and suggest a potential underlying mechanism involving altered OT signalling. Importantly, there was no desensitization of the feeding response over the treatment period and no apparent deleterious health effects, indicating that targeting the relaxin-3-RXFP3 system may be an effective long-term therapy for eating disorders.
Abstract Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, ...mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor; RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R385-101 ). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an 125 I-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.
Background and Purpose
In a recently conducted phase III clinical trial, RELAX‐AHF, serelaxin infusion over 48 h improved short‐ and long‐term clinical outcomes in patients with acute heart failure. ...In this study we used human primary cells from the umbilical vasculature to better understand the signalling mechanisms activated by serelaxin.
Experimental Approach
We examined the acute effects of serelaxin on signal transduction mechanisms in primary human umbilical vascular cells and its chronic actions on markers of cardiovascular function and disease.
Key Results
The RXFP1 receptor, the cognate serelaxin receptor, was expressed at the cell surface in HUVECs and human umbilical vein smooth muscle cells (HUVSMCs), human umbilical artery smooth muscle cells (HUASMCs) and human cardiac fibroblasts (HCFs), but not human umbilical artery endothelial cells. In HUVECs and HUVSMCs, serelaxin increased cAMP, cGMP accumulation and pERK1/2, and the concentration–response curves (CRCs) were bell‐shaped. Similar bell‐shaped CRCs for cGMP and pERK1/2 were observed in HCFs, whereas in HUASMCs, serelaxin increased cAMP, cGMP and pERK1/2 with sigmoidal CRCs. Gαi/o and lipid raft disruption, but not Gαs inhibition, altered the serelaxin CRC for cAMP and cGMP accumulation in HUVSMC but not HUASMC. Longer term serelaxin exposure increased the expression of neuronal NOS, VEGF, ETβ receptors and MMPs (gelatinases) in RXFP1 receptor‐expressing cells.
Conclusions and Implications
Serelaxin caused acute and chronic changes in human umbilical vascular cells that were cell background dependent. Bell‐shaped CRCs that were observed only in venous cells and fibroblasts involved Gαi/o located within membrane lipid rafts.
Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous ...non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.
•Production of H2O2 can lead to oxidative stress and reduce sperm fertility.•We examine the effect of two antioxidants on rabbit sperm during chilled storage.•The addition of 1mM, 6mM or 12mM of ...methionine does not affect the level of H2O2.•Quercetin protects sperm against oxidative stress by reducing H2O2 production.
Reactive oxygen species, such as hydrogen peroxide, H2O2, can reduce sperm quality during storage. This study evaluated the effect of methionine and quercetin on rabbit sperm quality during liquid storage over 96h. Semen was collected from adult bucks (n=4) and pooled following evaluation. In Experiment 1, pooled ejaculates were diluted with a Tris extender supplemented with methionine (1, 6 or 12mM), quercetin (50 or 200μM) or no antioxidant (control) and then subdivided for storage at 5°C or 15°C. Sperm quality was assessed by CASA (total motility TM) and flow cytometry (viability, acrosome integrity and H2O2 production) at 0, 48, 72 and 96h. Experiments were replicated three times. Motility was significantly higher in control samples and lowest following dilution with 200μM quercetin, irrespective of storage temperature. Storage at 15°C improved viability and acrosome integrity compared with 5°C, but produced significantly more H2O2 at 72 and 96h in sperm diluted with methionine or no antioxidant. Quercetin-supplemented spermatozoa exhibited lower levels of H2O2 at both storage temperatures for all incubation times (P<0.05). In Experiment 2, the concentration of quercetin (0, 25, 50, 100 and 200μM) was investigated with additional quality parameters; lipid peroxidation and DNA integrity. All concentrations of quercetin reduced H202 and lipid peroxidation during storage at 15°C, but were not beneficial for TM, viability, acrosome or DNA integrity. Only supplementation with 100 and 200μM quercetin resulted in similar H202 levels at 5°C and 15°C (P>0.05). Overall, quercetin-supplementation to sperm medium provided protection against oxidative stress in 15°C-stored rabbit spermatozoa over 96h.
Despite being developed in its present form 20 years ago, sex-sorting of mammalian sperm is still a work in progress. While relatively successful in cattle and sheep, the unique challenges of ...incorporating sex-sorted sperm into pig production have not yet been overcome. Generally speaking, boar sperm survive freeze-thawing less well and are required in larger numbers for insemination, while in vitro embryo production of pig embryos is less successful compared to other domestic species
Niemann H, Rath D. Progress in reproductive biotechnology in swine. Theriogenology 2001;56:1291–1304. Due to the large number of sperm required for artificial insemination in pigs, a technique of storing sperm after sorting must be developed while adequate numbers of sperm are allocated into X- or Y-chromosome-bearing enriched pools. Cryopreservation is perhaps the ideal method of storage between sorting and insemination, as it allows unlimited time to build up a sperm bank, whereas liquid-storage requires the use of sperm within days of sorting.
The limited number of studies investigating the survivability of sex-sorted, frozen-thawed boar sperm have produced promising in vitro results but poor in vivo outcomes. Before fertility can be improved, the causes of any damage to sperm function during the sex-sorting and freeze-thawing procedures must be more fully understood. Once defined, the source of damage may be minimised and this would lead to increased success rates after in vivo application of sex-sorted, frozen-thawed boar sperm.
This study compared protocols for cryopreservation of ejaculated, papain-treated alpaca spermatozoa. This included different concentrations of egg yolk (EY; 5, 10 or 15%) and glycerol (2, 5 or 10%), ...diluent types (SHOTOR, lactose, skim milk or INRA-96™), freeze rates (2, 4 or 8 cm above liquid nitrogen; LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Spermatozoa were assessed pre-freeze and 0, 30, 60 and 90 min post-thaw. Forty-one hembras were inseminated with either fresh, papain-treated or frozen-thawed spermatozoa. Motility was affected by EY concentration (P < 0.001), diluent type (P < 0.001), freeze rate (P = 0.003) and storage vessel (P = 0.001). Viability was affected by EY concentration (P < 0.001), diluent type (P < 0.001), storage vessel (P = 0.002) and thaw rate (P = 0.03). For artificial insemination (AI), semen was diluted 1:3 in a lactose-based diluent, with 5% EY and glycerol. Freezing was in 0.5 mL straws, 2 cm above LN for 4 min then thawing at 37 °C for 1 min. Pregnancy rates of those ovulated (n = 26) were not different (1/5 fresh, 1/4 papain-treated, 0/17 frozen-thawed; P = 0.10). Pregnancy can be achieved after AI with papain-treated spermatozoa. Further work is needed to determine the optimal dose, timing and location for insemination.
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