Polygenic risk assessment reveals pleiotropy between sarcoidosis and inflammatory disorders in the context of genetic ancestry.

Sarcoidosis is a complex disease of unknown etiology characterized by the presence of granulomatous inflammation. Though various immune system pathways have been implicated in disease, the relationship between the genetic determinants of sarcoidosis and other inflammatory disorders has not been characterized. Herein, we examined the degree of genetic pleiotropy common to sarcoidosis and other inflammatory disorders to identify shared pathways and disease systems pertinent to sarcoidosis onset. To achieve this, we quantify the association of common variant polygenic risk scores from nine complex inflammatory disorders with sarcoidosis risk. Enrichment analyses of genes implicated in pleiotropic associations were further used to elucidate candidate pathways. In European-Americans, we identify significant pleiotropy between risk of sarcoidosis and risk of asthma (R2=2.03%; P=8.89 × 10-9), celiac disease (R2=2.03%; P=8.21 × 10-9), primary biliary cirrhosis (R2=2.43%; P=2.01 × 10-10) and rheumatoid arthritis (R2=4.32%; P=2.50 × 10-17). These associations validate in African Americans only after accounting for the proportion of genome-wide European ancestry, where we demonstrate similar effects of polygenic risk for African-Americans with the highest levels of European ancestry. Variants and genes implicated in European-American pleiotropic associations were enriched for pathways involving interleukin-12, interleukin-27 and cell adhesion molecules, corroborating the hypothesized immunopathogenesis of disease.

Effect of superstimulation protocols on nuclear maturation and distribution of lipid droplets in bovine oocytes.

Our objective was to study the effect of superstimulation protocols on nuclear maturation of the oocyte and the distribution of lipid droplets in the ooplasm. Heifers (n=4 each group) during the luteal phase were either treated with FSH for 4 days (Short FSH), FSH for 4 days followed by 84h of gonadotropin free period (FSH Starvation) or for 7 days (Long FSH) starting from the day of wave emergence. In all groups, LH was given 24h after induced luteolysis (penultimate day of FSH) and cumulus-oocyte complexes were collected 24h later. Oocytes were stained for nuclear maturation (Lamin/chromatin) and lipid droplets (Nile red). The Long FSH group had a greater proportion of mature oocytes (metaphase II) compared with heifers in the Short FSH and FSH Starvation groups (59/100 vs 5/23 and 2/25, respectively; P<0.01). On average across all groups, oocytes contained 22pL of lipids (3.3% of ooplasm volume) distributed as 3000 droplets. Average volume of individual lipid droplets was higher in the FSH Starvation (11.5±1.5 10(-3) pL, P=0.03) compared with the Short and Long FSH groups (7.2±0.6 10(-3) and 8.0±0.8 10(-3) pL, respectively). In conclusion, both FSH Starvation and Short FSH treatments yielded a lower proportion of mature oocytes compared with the Long FSH treatment. Furthermore, FSH starvation led to an accumulation of larger lipid droplets in the ooplasm, indicating atresia. Our results indicate that a longer superstimulation period in beef cattle yields higher numbers and better-quality oocytes.

Effects of pipothiazine palmitate on handling stress and on the characteristics of semen collected by electroejaculation in bison (Bison bison) bulls.

Handling North American bison can pose risk to the handler and evoke stress in the animal. Moreover, this induced stress might affect qualities of semen collected by electroejaculation. The objective of this study was to investigate if a long acting neuroleptic tranquilizer (LAN) would reduce the stress of bison and thereby improve the quality of electroejaculated semen. Eight experimental replicates were conducted between May and November. In each replicate, the same six bison bulls were randomly assigned into LAN-treated (n=3) and non-treated control (n=3) groups. Pipothiazine palmitate (Piportil L4) was administered intramuscularly as a single dose of 100 mg in replicates 1-4 or 200 mg in replicates 5-8. Within each replicate, semen was collected by electroejaculation at 4, 6, 11 and 13 days post treatment. Behavioral parameters, sperm morphology and motility parameters were analyzed. A blood sample was collected before each electroejaculation and serum concentrations of testosterone, cortisol and corticosterone were determined. Treatment bulls with 100 mg of Piportil L4 reduced the restraint time and the struggling of bison bulls during handling compared to the control group (P<0.05). Semen motility parameters and serum concentrations of testosterone, cortisol and corticosterone were not significantly affected when 100mg of the LAN was administered (P>0.05). However, giving 200 mg of Piportil L4 reduced the restraint time of bison bulls and the duration of semen collection (P<0.05). Also, this treatment improved total and progressive sperm motilities when compared to the respective controls (P<0.05). Interestingly, serum concentration of corticosterone, as an endocrine stress indicator, was decreased after administration of 200mg of Pipothiazine palmitate, while testosterone concentrations were increased compared to those values in untreated control bulls (corticosterone: 0.10±0.01 compared with 0.15±0.02 ng/mL; testosterone: 9.11±1.68 compared with 5.33±0.74 ng/mL; P<0.05). In conclusion, this study demonstrated that a treatment dose of 200mg of Piportil L4 can decrease the behavioral and endocrine stress responses in bison bulls, which indirectly increasing testosterone concentrations and improving semen quality.

A strategy for improvement of postthaw quality of bison sperm.

The objective was to improve the postthaw quality of bison semen using zwitterion (ZI)-based extenders, glycerol addition at a lower temperature (4 °C), adding reduced glutathione (GSH) in extender, or treating bison sperm with cholesterol-loaded cyclodextrin (CLC) before freezing. Postthaw sperm motility and structural characteristics were analyzed using a computer-assisted sperm analyzer and flow cytometer respectively, at 0 and 3 hours postthaw incubation at 37 °C. In experiment 1, each ejaculate (N = 11) was diluted in Triladyl extender (control) or in ZI extenders (Tes-Tris or HEPES-Tris). In addition, glycerol in semen was added either at 37 °C or 4 °C before cryopreservation. Extenders had no significant effect on postthaw sperm motilities at 0 hour. However, sperm velocity parameters were higher (P < 0.05) in ZI extenders than in Triladyl. Sperm population with intact plasma membrane (IPM) and acrosomes (IACR) were higher in Triladyl than in ZI extenders (P < 0.05). Postthaw sperm total and progressive motilities, average path velocity, straight-line velocity, IPM, and IPM-IACR did not improve with the addition of glycerol at 4 °C. In experiment 2, semen was diluted (50 × 10(6) sperm per mL) in Triladyl extender containing 0 (control), 0.5, 1.0, or 2.0 mM GSH (an antioxidant) at 37 °C. Postthaw sperm motility and structural characteristics at 0 hour and percentage declined after 3 hour incubation, but did not differ because of GSH in the extender (P > 0.05). In experiment 3, fresh bison sperm (100 × 10(6) sperm in 1 mL) were pretreated with 0, 1, 2, or 3 mg/mL of CLC at 22 °C for 15 minutes and diluted to 50 × 10(6) sperm per mL in Tris-citric acid-egg yolk-glycerol extender before cryopreservation. The CLC pretreated sperm had higher (P < 0.05) postthaw total and progressive motilities, IPM, and IACR at 0 hour and less percentage of decline in these characteristics after 3 hour postthaw incubation. In conclusion, zwitterion extenders (Tes-Tris and HEPES-Tris), temperatures of glycerol addition, and GSH in extender did not significantly improve postthaw quality of bison sperm. However, pretreatment with CLC significantly improved postthaw quality of bison sperm, which should enhance its use in assisted reproductive technologies.

A functional haplotype of UBE2L3 confers risk for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations characterized by the development of pathogenic autoantibodies manifesting in inflammation of target organs such as the kidneys, skin and joints. Genome-wide association studies have identified genetic variants in the UBE2L3 region that are associated with SLE in subjects of European and Asian ancestry. UBE2L3 encodes an ubiquitin-conjugating enzyme, UBCH7, involved in cell proliferation and immune function. In this study, we sought to further characterize the genetic association in the region of UBE2L3 and use molecular methods to determine the functional effect of the risk haplotype. We identified significant associations between variants in the region of UBE2L3 and SLE in individuals of European and Asian ancestry that exceeded a Bonferroni-corrected threshold (P<1 × 10(-4)). A single risk haplotype was observed in all associated populations. Individuals harboring the risk haplotype display a significant increase in both UBE2L3 mRNA expression (P=0.0004) and UBCH7 protein expression (P=0.0068). The results suggest that variants carried on the SLE-associated UBE2L3 risk haplotype influence autoimmunity by modulating UBCH7 expression.

Role of MYH9 and APOL1 in African and non-African populations with lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and organ damage. Lupus nephritis (LN) is one of the most severe manifestations of SLE. Multiple studies reported associations between renal diseases and variants in the non-muscle myosin heavy chain 9 (MYH9) and the neighboring apolipoprotein L 1 (APOL1) genes. We evaluated 167 variants spanning MYH9 for association with LN in a multiethnic sample. The two previously identified risk variants in APOL1 were also tested for association with LN in European-Americans (EAs) (N = 579) and African-Americans (AAs) (N = 407). Multiple peaks of association exceeding a Bonferroni corrected P-value of P < 2.03 × 10(-3) were observed between LN and MYH9 in EAs (N = 4620), with the most pronounced association at rs2157257 (P = 4.7 × 10(-4), odds ratio (OR) = 1.205). A modest effect with MYH9 was also detected in Gullah (rs8136069, P = 0.0019, OR = 2.304). No association between LN and MYH9 was found in AAs, Asians, Amerindians or Hispanics. This study provides the first investigation of MYH9 in LN in non-Africans and of APOL1 in LN in any population, and presents novel insight into the potential role of MYH9 in LN in EAs.

Infertility in a beef bull due to a failure in the capacitation process.

The objective of this case report was to identify the cause of apparent idiopathic infertility in a Red Angus (beef) bull. Semen was collected by electroejaculation and submitted to a series of assays, including evaluation of sperm motility by computer-assisted sperm analysis (CASA), sperm morphology and DNA integrity, semen cryopreservation, AI, IVF, induction of the acrosome reaction, and determination of the level of sperm proteins associated with bull fertility potential. Total (92 ± 2%) and progressive (79 ± 4%) sperm motility; sperm concentration (1647 ± 429 × 10(6) sperm/mL); proportions of morphologically normal sperm (83 ± 6%) and DNA integrity (96 ± 2), and acrosome-intact sperm (64 ± 4%) exceeded minimum acceptable values. Frozen sperm had good total (58.7 ± 6.7%) and progressive (43.9 ± 9.2%) motility immediately after thawing. However, AI of 16 heifers resulted in no pregnancies and blastocyst production rate (following IVF using sperm from this infertile bull) was nearly identical to that produced using dead sperm (a control of parthenogenesis; 2 ± 2 and 2 ± 3%; respectively P < 0.05). Treatment with a calcium ionophore (A23187) failed to induce the acrosome reaction in sperm from the infertile bull (P < 0.05). Evaluation of several proteins associated with the fertility potential of bulls revealed that the level of Binder Sperm Protein-1 (BSP1), known to be associated with the capacitation process, was much greater on sperm from the infertile bull compared to that of his sire. In conclusion, we inferred that the idiopathic infertility in this bull was caused by a failure to complete the capacitation process.

Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality.

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(®) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl® extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl® or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl®, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl®. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl® at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.

Recent insights into the genetic basis of systemic lupus erythematosus.

Genetic variation was first shown to be important in systemic lupus erythematosus (SLE or lupus) in the 1970s with associations in the human leukocyte antigen region. Almost four decades later, and with the help of increasingly powerful genetic approaches, more than 25 genes are now known to contribute to the mechanisms that predispose individuals to lupus. Over half of these loci have been discovered in the past 2 years, underscoring the extraordinary success of genome-wide association approaches in SLE. Well-established risk factors include alleles in the major histocompatibility complex region (multiple genes), IRF5, ITGAM, STAT4, BLK, BANK1, PDCD1, PTPN22, TNFSF4, TNFAIP3, SPP1, some of the Fcgamma receptors, and deficiencies in several complement components, including C1q, C4 and C2. As reviewed here, many susceptibility genes fall into key pathways that are consistent with previous studies implicating immune complexes, host immune signal transduction and interferon pathways in the pathogenesis of SLE. Other loci have no known function or apparent immunological role and have the potential to reveal novel disease mechanisms. Certainly, as our understanding of the genetic etiology of SLE continues to mature, important new opportunities will emerge for developing more effective diagnostic and clinical management tools for this complex autoimmune disease.

Peripheral blood gene expression profiling in Sjögren's syndrome.

Sjögren's syndrome (SS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands. The affected cases commonly present with oral and ocular dryness, which is thought to be the result of inflammatory cell-mediated gland dysfunction. To identify important molecular pathways involved in SS, we used high-density microarrays to define global gene expression profiles in the peripheral blood. We first analyzed 21 SS cases and 23 controls, and identified a prominent pattern of overexpressed genes that are inducible by interferons (IFNs). These results were confirmed by evaluation of a second independent data set of 17 SS cases and 22 controls. Additional inflammatory and immune-related pathways with altered expression patterns in SS cases included B- and T-cell receptor, insulin-like growth factor-1, granulocyte macrophage-colony stimulating factor, peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha and PI3/AKT signaling. Exploration of these data for relationships to clinical features of disease showed that expression levels for most interferon-inducible genes were positively correlated with titers of anti-Ro/SSA (P<0.001) and anti-La/SSB (P<0.001) autoantibodies. Diagnostic and therapeutic approaches targeting interferon-signaling pathway may prove most effective in the subset of SS cases that produce anti-Ro/SSA and anti-La/SSB autoantibodies. Our results strongly support innate and adaptive immune processes in the pathogenesis of SS, and provide numerous candidate disease markers for further study.

Meta-analysis and imputation identifies a 109 kb risk haplotype spanning TNFAIP3 associated with lupus nephritis and hematologic manifestations.

TNFAIP3 encodes the ubiquitin-modifying enzyme, A20, a key regulator of inflammatory signaling pathways. We previously reported association between TNFAIP3 variants and systemic lupus erythematosus (SLE). To further localize the risk variant(s), we performed a meta-analysis using genetic data available from two Caucasian case-control datasets (1453 total cases, 3381 total control subjects) and 713 SLE trio families. The best result was found at rs5029939 (P=1.67 x 10(-14), odds ratio=2.09, 95% confidence interval 1.68-2.60). We then imputed single nucleotide polymorphisms (SNPs) from the CEU Phase II HapMap using genotypes from 431 SLE cases and 2155 control subjects. Imputation identified 11 SNPs in addition to three observed SNPs, which together, defined a 109 kb SLE risk segment surrounding TNFAIP3. When evaluating whether the rs5029939 risk allele was associated with SLE clinical manifestations, we observed that heterozygous carriers of the TNFAIP3 risk allele at rs5029939 have a twofold increased risk of developing renal or hematologic manifestations compared to homozygous non-risk subjects. In summary, our study strengthens the genetic evidence that variants in the region of TNFAIP3 influence risk for SLE, particularly in patients with renal and hematologic manifestations, and narrows the risk effect to a 109 kb DNA segment that spans the TNFAIP3 gene.

Banking North American buffalo semen.

The purpose of this study was to develop a procedure to collect and preserve semen from wood bison (Bison bison athabascae) and plains bison (Bison bison bison). Semen samples from three wood and three plains bison bulls were collected by electroejaculation from June through October. In addition, sperm was collected from the cauda epididymis of seven plains bison. Semen was cryopreserved using two commercially available cryopreservation media, an egg yolk-based medium (Triladyl), and a medium free of products of animal origin (Andromed). Sperm morphology and motility were recorded on fresh and post-thawed semen samples. Total sperm motility was not different between plains and wood bison for the months of June (50%), July (69%) and October (54%). However, total sperm motility for wood bison was higher (P<0.05) than plains bison for the months of August and September (August: 80% vs 55%; September: 73% vs 40%). Plains and wood bison did not differ in mean total and mean progressive motility (35 and 15%, respectively) of frozen-thawed sperm samples. The post-thaw motility of Triladyl-treated sperm was higher (P<0.05) than Andromed-treated sperm (35% vs 13%, respectively). Interestingly, post-thawed epididymal spermatozoa had higher total motility (P<0.05) than post-thawed electroejaculated sperm when cryopreserved with a medium free of products of animal origin (Andromed; 35% vs 9%, respectively). In conclusion, we used electroejaculation to collect high quality bison semen, and cryopreserved it for future needs.

Mutagenesis-generated mouse models of human infertility with abnormal sperm.

BACKGROUND: The aetiology of human male fertility, with impairment of sperm number, motility and morphology (oligoasthenoteratozoospermia), has been difficult to understand, partly for lack of animal models. METHODS: An ethylnitrosourea (ENU) mutagenesis strategy has been successful in producing heritable gene mutations with phenotypes similar to human male infertility, and here, we describe three independent ENU-induced mutations that cause a phenotype of oligoasthenoteratozoospermia in mice. RESULTS: The loci identified by these three mutations are designated swm2, repro2 and repro3. All mutant males were characterized by low sperm concentration, poor sperm morphology and negligible motility, but the infertile males were apparently normal in other respects. Sperm from mutant males failed to fertilize oocytes in vitro. Ultrastructural analyses revealed varied abnormalities apparent in both testicular spermatids and epididymal sperm. Genetic mapping placed the swm2 gene on chromosome 7, the repro2 gene on chromosome 5 and the repro3 gene on chromosome 10. CONCLUSION: The single-gene mutations caused complex and non-specific sperm pathologies, a point with important implications for managing cases of human male infertility. The ultimate identification of the loci for the mutations causing these phenotypes will clarify aetiology of complex syndromes of infertility with sperm abnormalities consistent with oligoasthenoteratozoospermia.

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.

Cryopreservation alters the levels of the bull sperm surface protein P25b.

Fertility of frozen-thawed bull sperm is reduced by cryopreservation. Freezing-thawing procedures can result in as much as a sevenfold fertility decrease. Sperm mortality and loss of motility do not fully explain the reduced fertility of cryopreserved semen; they may be partially explained by the loss of sperm surface proteins, which are necessary for fertilization. We have previously identified P25b, a sperm surface protein, which is associated with the fertility index of bulls used for artificial insemination. Using Western blotting techniques, we have evaluated P25b levels before and after cryopreservation of bull spermatozoa in extenders based on either egg yolk or milk. Long storage periods (28 days) in liquid nitrogen results in a threefold decrease of P25b levels associated with cryopreserved versus fresh spermatozoa. Over a short storage period (3-7 days), a stable P25b level was observed on spermatozoa cryopreserved in extender containing either egg yolk or milk. A decrease in P25b levels associated with spermatozoa was observed after 5 days of storage in egg yolk extender, whereas a significant decrease was observed after 14 days of sperm storage in milk extender (P < .05). Therefore, the loss of P25b may be responsible, at least in part, for the decrease in fertility following the freezing-thawing procedure of bull semen. Moreover, the cryopreservation extender used may have different effects on the loss of sperm surface proteins after even brief storage periods in liquid nitrogen. Considering that a sperm protein similar to P25b exists in humans (P34H), these results may have significant clinical applications in which frozen semen is used.

Comparison of optokinetic scene effects on the somatogyral illusion.

The objective of this study was to determine during rotation the relative importance of a scene in achieving "visual dominance" over non-visual vestibular orientation inputs, e.g., otolith and semicircular canals. Five visual scenes were presented, while rotating the subject (at three angular acceleration rates), to obtain the vestibular and optokinetic nystagmus and perception of rotation. The onset time and direction of rotation, as well as EOG and chair velocity were recorded. Adaptation times during rotation at constant angular velocity and during post-rotation were also recorded. Analysis of perceived times (onset, adaptation, and post-rotatory adaptation) with the EOG slow-phase velocity at respectively perceived times were analyzed using the SAS procedures Analysis of Variance (ANOVA) and General Linear Models (GLM). Basic hypotheses for the study were: "There are no differences in latencies for either onset time (L1), adaptation to rotation (L2), or adaptation to post rotatory motion (L4), between treatment conditions, or between Groups of subjects, or between acceleration rates, between constant rotation velocities, or between direction of rotation." From the results, it was concluded that pilots are highly visually dependent; additionally, if there is a sensory conflict, a larger percentage of individuals (pilots) will follow visual perceptions even if the demands of the aerial environment and our perception is incorrect. However, in sensory conflict conditions, with degraded visual scene individuals including pilots will revert to vestibular perception rather than visual perception (less visually coupled), approaching percentages as noted with a dark environment.