Relaxin family peptides and their receptors.

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.

Relaxin-3 inputs target hippocampal interneurons and deletion of hilar relaxin-3 receptors in "floxed-RXFP3" mice impairs spatial memory.

Hippocampus is innervated by γ-aminobutyric acid (GABA) "projection" neurons of the nucleus incertus (NI), including a population expressing the neuropeptide, relaxin-3 (RLN3). In studies aimed at gaining an understanding of the role of RLN3 signaling in hippocampus via its Gi/o -protein-coupled receptor, RXFP3, we examined the distribution of RLN3-immunoreactive nerve fibres and RXFP3 mRNA-positive neurons in relation to hippocampal GABA neuron populations. RLN3-positive elements were detected in close-apposition with a substantial population of somatostatin (SST)- and GABA-immunoreactive neurons, and a smaller population of parvalbumin- and calretinin-immunoreactive neurons in different hippocampal areas, consistent with the relative distribution patterns of RXFP3 mRNA and these marker transcripts. In light of the functional importance of the dentate gyrus (DG) hilus in learning and memory, and our anatomical data, we examined the possible influence of RLN3/RXFP3 signaling in this region on spatial memory. Using viral-based Cre/LoxP recombination methods and adult mice with a floxed Rxfp3 gene, we deleted Rxfp3 from DG hilar neurons and assessed spatial memory performance and affective behaviors. Following infusions of an AAV(1/2) -Cre-IRES-eGFP vector, Cre expression was observed in DG hilar neurons, including SST-positive cells, and in situ hybridization histochemistry for RXFP3 mRNA confirmed receptor depletion relative to levels in floxed-RXFP3 mice infused with an AAV(1/2) -eGFP (control) vector. RXFP3 depletion within the DG hilus impaired spatial reference memory in an appetitive T-maze task reflected by a reduced percentage of correct choices and increased time to meet criteria, relative to control. In a continuous spontaneous alternation Y-maze task, RXFP3-depleted mice made fewer alternations in the first minute, suggesting impairment of spatial working memory. However, RXFP3-depleted and control mice displayed similar locomotor activity, anxiety-like behavior in light/dark box and elevated-plus maze tests, and learning and long-term memory retention in the Morris water maze. These data indicate endogenous RLN3/RXFP3 signaling can modulate hippocampal-dependent spatial reference and working memory via effects on SST interneurons, and further our knowledge of hippocampal cognitive processing. © 2017 Wiley Periodicals, Inc.

Supplementation of culture medium with L-carnitine improves the development and cryotolerance of in vitro-produced porcine embryos.

Porcine oocytes and embryos contain substantial amounts of lipid, with little known regarding its metabolic role during development. This study investigated the role of lipid metabolism and the interaction between carbohydrate and lipid substrates in porcine embryos. Following in vitro fertilisation, presumptive zygotes were transferred to culture medium supplemented with L-carnitine, a co-factor required for the metabolism of fatty acids. In porcine zygote medium-3 (PZM-3), which contains pyruvate and lactate, 3mM L-carnitine was the only dose that improved cleavage rates compared with the control. In the absence of carbohydrates, all doses of L-carnitine from 1.5 to 12mM increased cleavage rates compared with the control. Culture in a PZM-3-based sequential media system (Days 0-3: pyruvate and lactate; Days 4-7: glucose) significantly increased blastocyst cell numbers compared with culture in standard PZM-3. Supplementing PZM-3 with 3mM L-carnitine produced blastocysts with cell numbers equivalent to those obtained in the sequential media system. After vitrification, the post-warming survival rates of blastocysts obtained in media supplemented with 3mM L-carnitine were significantly greater than those of blastocysts obtained in standard PZM-3. In conclusion, L-carnitine supplementation improved embryo development when the medium contained pyruvate and lactate or was lacking carbohydrates completely, indicating a role for fatty-acid metabolism when the embryo's requirements for carbohydrates are not adequately met.

Variation in seminal plasma alters the ability of ram spermatozoa to survive cryopreservation.

Variation in the effect of seminal plasma on sperm function and fertility has been hypothesised to be due to differences between males and their seminal plasma composition. The freezing resilience of individual rams (n=17) was investigated to characterise inter-male variation. This was determined by measuring the degree of change in motility induced by cryopreservation (Experiment 1). Experiment 2 examined the effect of pooled seminal plasma from rams identified as having high or low resilience to freezing on the cryosurvival of washed spermatozoa from either high (n=3) or low (n=3) sperm freezing resilience rams. Immediately after thawing and throughout the incubation period (0-4h), spermatozoa from high-resilience rams frozen with high-resilience seminal plasma demonstrated superior motility to spermatozoa from high-resilience rams frozen with low-resilience seminal plasma (P<0.001). Similarly, spermatozoa from low-resilience rams frozen with high-resilience seminal plasma exhibited higher motility than spermatozoa from low-resilience rams frozen with low-resilience seminal plasma immediately after thawing (0h; P<0.001). The present study shows that variation in freezing resilience of ram spermatozoa is related to the source and composition of the seminal plasma.

Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications.

Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular 'omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual's genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in systems genomics has to be accompanied by an equally strong effort to develop next-generation DNA-sequencing and next-generation drug screening and design platforms. In that context lab-on-a-chip devices utilizing nanopore- and nanochannel based resistive pulse-sensing technology for DNA-sequencing and protein screening applications occupy a key role. This paper describes the status quo of resistive pulse sensing technology for these two application areas with a special focus on current technology trends and challenges ahead.

Effects of varying doses of ß-nerve growth factor on the timing of ovulation, plasma progesterone concentration and corpus luteum size in female alpacas (Vicugna pacos).

Ovulation in camelids is induced by the seminal plasma protein ovulation-inducing factor (OIF), recently identified as ß-nerve growth factor (ß-NGF). The present study measured the total protein concentration in alpaca seminal plasma using a bicinchoninic acid (BCA) protein quantification assay and found it to be 22.2±2.0mgmL(-1). To measure the effects of varying doses of ß-NGF on the incidence and timing of ovulation, corpus luteum (CL) size and plasma progesterone concentration, 24 female alpacas were synchronised and treated with either: (1) 1mL 0.9% saline (n=5); (2) 4µg buserelin (n=5); (3) 1mg ß-NGF protein (n=5); (4) 0.1mg ß-NGF (n=5); or (5) 0.01mg ß-NGF (n=4). Females were examined by transrectal ultrasonography at 1-2-h intervals between 20 and 45h after treatment or until ovulation occurred, as well as on Day 8 to observe the size of the CL, at which time blood was collected to measure plasma progesterone concentrations. Ovulation was detected in 0/5, 5/5, 5/5, 3/5 and 0/4 female alpacas treated with saline, buserelin, 1, 0.1 and 0.01mg ß-NGF, respectively. Mean ovulation interval (P=0.76), CL diameter (P=0.96) and plasma progesterone concentration (P=0.96) did not differ between treatments. Mean ovulation interval overall was 26.2±1.0h. In conclusion, buserelin and 1mg ß-NGF are equally effective at inducing ovulation in female alpacas, but at doses ≤0.1mg, ß-NGF is not a reliable method for the induction of ovulation.

The Effect of Sperm Concentration and Storage Vessel on Quercetin-Supplemented Rabbit Semen During Chilled Storage.

Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris-based extender supplemented with 100 µm quercetin to a concentration of 15, 30 or 60 × 10(6)  spermatozoa/ml, packaged into plastic tubes or 0.5-ml straws and stored at 15°C. Sperm quality was assessed by computer-assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H2 O2 production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10(6)  spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD-DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10(6)  spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10(6)  spermatozoa/ml may provide a suitable balance between motility and H2 O2 production to best maintain overall sperm function and should be evaluated in a large-scale AI trial.

Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa.

Seminal plasma purportedly plays a critical role in reproduction, but epididymal spermatozoa are capable of fertilisation following deposition in the uterus, calling into question the biological requirement of this substance. Through a combination of direct observation of spermatozoa in utero using probe-based Confocal Laser Endomicroscopy, in vivo assessment of sperm fertility and in vitro analysis of various sperm functional parameters, this study investigated the role of seminal plasma in spermatozoa transit through the cervix of the ewe. Following deposition in the cervical os, epididymal spermatozoa previously exposed to seminal plasma displayed an enhanced ability to traverse the cervix as evidenced by both significantly higher pregnancy rates and numbers of spermatozoa observed at the utero-tubal junction when compared with epididymal spermatozoa not previously exposed to seminal plasma. The beneficial effect of seminal plasma on sperm transport was clearly localised to transit through the cervix as pregnancy rates of spermatozoa deposited directly into the uterus were unaffected by exposure to seminal plasma. This phenomenon was not explained by changes to sperm motion characteristics, as seminal plasma had no effect on the motility, kinematic parameters or mitochondrial membrane potential of spermatozoa. Rather, in vitro testing revealed that seminal plasma improved the ability of epididymal spermatozoa to penetrate cervical mucus recovered from ewes in oestrus. These results demonstrate that the survival and transport of ram spermatozoa through the cervix of the ewe is not linked to their motility or velocity but rather the presence of some cervical penetration trait conferred by exposure to seminal plasma.

Modulation of feeding by chronic rAAV expression of a relaxin-3 peptide agonist in rat hypothalamus.

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.

Birth of kids after artificial insemination with sex-sorted, frozen-thawed goat spermatozoa.

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.

Relaxin family peptide systems and the central nervous system.

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.

Antioxidant mechanisms and their benefit on post-thaw boar sperm quality.

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.

Flow-sorted ram spermatozoa are highly susceptible to hydrogen peroxide damage but are protected by seminal plasma and catalase.

To determine whether flow sorting increased the susceptibility of spermatozoa to reactive oxygen species (ROS), ram semen was either diluted with Tris medium (100 x 10(6) spermatozoa mL(-1); D) or highly diluted (10(6) spermatozoa mL(-1)) before being centrifuged (DC) at 750g for 7.5 min at 21 degrees C or flow-sorted (S) before cryopreservation. Thawed spermatozoa were resuspended in graded concentrations of hydrogen peroxide to induce oxidative stress. In Experiment 1, following exposure to 30 or 45 muM hydrogen peroxide (H(2)O(2)), the total motility (%) of DC (41.0 +/- 7.3 or 25.7 +/- 6.7, respectively) and S spermatozoa (33.8 +/- 6.3 or 20.1 +/- 6.3, respectively) was lower (P < 0.001) than that of D spermatozoa (58.7 +/- 5.6 or 44.5 +/- 6.7, respectively). In Experiment 2, supplementation of samples containing H(2)O(2) with catalase (150 IU mL(-1)) or seminal plasma proteins (4 mg protein per 10(8) spermatozoa) negated oxidative stress, resulting in comparable values to samples receiving no H(2)O(2)in terms of the proportion of spermatozoa with stable plasmalemma (as determined using merocyanine-540 and Yo-Pro-1) in the D and S groups, the proportion of viable, acrosome-intact spermatozoa (as determined by fluorescein isothiocyanate and propidium iodide staining) in the D group and the motility of control (undiluted) and S spermatozoa. Neither H(2)O(2) nor sperm type (i.e. D, DC or S) had any effect on intracellular concentrations of ROS. These results show that flow sorting increases the susceptibility of spermatozoa to ROS, but the inclusion of anti-oxidants or seminal plasma as part of the sorting protocol improves resistance to oxidative stress.

Development of procedures for sex-sorting frozen-thawed bovine spermatozoa.

Dairy bull sperm may be sex-sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well-managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex-sorting from frozen-thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex-sort and re-freeze frozen-thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm or BoviPure, followed by staining in one of three diluents (Androhep, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen-thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 +/- 0.6% vs 19.5 +/- 2.4%; p = 0.003) after centrifugation in PureSperm rather than BoviPure gradients. Sperm orientation (p < 0.05) and motility (69.9 +/- 3.0 vs 55.6 +/- 4.0; p < 0.001) were highest after staining in Androhep rather than in TALP buffer. Sperm were more motile (58.2 +/- 4.7 vs 38.7 +/- 3.5; p < 0.001) and had better acrosome integrity (74.3 +/- 2.9 vs 66.8 +/- 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen-thawed bull sperm to be sex-sorted with high resolution between the sexes, then re-frozen and thawed with retention of motility and acrosome integrity.

Effect of diluent type, cryoprotectant concentration, storage method and freeze/thaw rates on the post-thaw quality and fertility of cryopreserved alpaca spermatozoa.

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.

Liquid storage of dromedary camel semen in different extenders.

This study was conducted to assess the effects of commercial extenders and storage temperature on dromedary camel sperm quality during liquid preservation. In Experiment 1, ejaculates (n = five males; replicated seven times) were split and diluted with synthetic (OPTIXcell, EquiPlus, INRA96, Bioxcell or AndroMed; Experiment 1a) or egg-yolk based (Biladyl, Green buffer or Triladyl; Experiment 1b) extenders and stored for 48 h at 4 °C. In Experiment 2, split ejaculates (n = five males; replicated six times) were used to directly compare Green buffer, OPTIXcell and Triladyl extenders over 48 h of storage at 4 °C. Ejaculates collected in Experiment 3 (n = five males; replicated five times) were diluted with Green buffer or Triladyl before chilled storage for 48 h at 4 or 15 °C. Sperm kinematics, viability and acrosome integrity were assessed during liquid storage. In Experiment 1a, there was the greatest total sperm motility (TM) in the OPTIXcell group following 24 and 48 h of storage, while in Experiment 1b, there was the greatest TM after 48 h of storage with Triladyl and Green buffer. In Experiment 2, there were greater TM and viable acrosome intact spermatozoa in the Triladyl and Green buffer than with OPTIXcell group. In Experiment 3, there was a greater TM in the Triladyl than Green buffer group at 24 and 48 h of storage regardless of storage temperature (which had no effect on sperm quality). In conclusion, camel sperm have greater viability when preserved in liquid form for 48 h following dilution with Triladyl and storage at either 4 or 15 °C.

Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function.

The relaxin receptor, RXFP1, is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346, which are highly conserved across species. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors, although its role in modulating receptor function differs. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at individual or combinations of double and triple sites had little effect on relaxin binding. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling.

Adenovirus-mediated delivery of relaxin reverses cardiac fibrosis.

We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to beta(2)-adrenergic receptor (beta(2)AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in beta(2)AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.

Early pregnancy loss in sows after low dose, deep uterine artificial insemination with sex-sorted, frozen-thawed sperm.

Recent developments in reproductive technologies have enabled the production of piglets of a predetermined sex via non-surgical, low dose artificial insemination. The practical application of sex-sorting technology to the pig is made challenging by the large numbers of sperm required for successful insemination of sows. One way of overcoming the time required for sex-sorting may be to create a bank of cryopreserved, sex-sorted sperm, thus making available appropriate doses as sows require insemination. To date, little success has been achieved with non-surgical inseminations of sex-sorted boar sperm. This study attempted to achieve litters of a predetermined sex after a double insemination of sows with 160x10(6) sex-sorted, frozen-thawed sperm. Sows were synchronised and sperm were non-surgically inseminated into the proximal third of the uterine horn at 36 and 42 h after hCG administration. Sows inseminated with sex-sorted sperm achieved similar pregnancy rates to those receiving an equal dose of unsorted, frozen-thawed sperm. However, all sows conceiving after insemination with sex-sorted sperm returned to oestrus within 57 days of insemination. This was a higher rate of pregnancy loss than observed for sows inseminated with unsorted sperm (37.5%; P=0.031). A combination of low sperm numbers and potentially compromised developmental capability of embryos derived from sex-sorted sperm may have resulted in this early stage loss of pregnancy.

Field fertility of frozen-thawed boar sperm at low doses using non-surgical, deep uterine insemination.

The lowest dose of frozen-thawed boar sperm used for deep uterine artificial insemination (DUI) of sows has been 100x10(6). A three stage field study was performed to establish to what level the dose of frozen-thawed sperm used for DUI could be reduced without adversely affecting the fertility of the sow. In stage 1, 15 sows were inseminated twice with 1000x10(6) fresh or frozen-thawed sperm at 24 and 36 h post-detection of oestrus. In stage 2, 262 sows were inseminated with 62.5, 250 or 1000x10(6) fresh or frozen-thawed sperm at 24, 36, or 24 and 36 h after detection of oestrus. Stage 3 involved post mortem investigation of the uterine lining to assess damage caused by insertion of the insemination catheter. All sows inseminated in stage 1 of the study farrowed. In stage 2, the non-return (NRR) and farrowing rates of each group were compared to a control double cervical insemination of 3250x10(6) fresh sperm. As few as 62.5x10(6) fresh sperm could be deposited at a single insemination without reduction in NRR or farrowing rates compared with the control group. A double DUI with 250x10(6) frozen-thawed sperm was required before fertility was equivalent to the controls. Investigation of the uterine lining after insertion of the DUI catheter revealed evidence of bleeding, warranting further investigation of the viability of widespread use of the Firflex catheter, despite the promising fertility achieved here with low doses of spermatozoa.