Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Maternal blood transcriptome as a sensor of fetal organ maturation at the end of organogenesis in cattle†.

Harnessing information from the maternal blood to predict fetal growth is attractive yet scarcely explored in livestock. The objectives were to determine the transcriptomic modifications in maternal blood and fetal liver, gonads, and heart according to fetal weight and to model a molecular signature based on the fetal organs allowing the prediction of fetal weight from the maternal blood transcriptome in cattle. In addition to a contemporaneous maternal blood sample, organ samples were collected from 10 male fetuses at 42 days of gestation for RNA-sequencing. Fetal weight ranged from 1.25 to 1.69 g (mean = 1.44 ± 0.15 g). Clustering data analysis revealed clusters of co-expressed genes positively correlated with fetal weight and enriching ontological terms biologically relevant for the organ. For the heart, the 1346 co-expressed genes were involved in energy generation and protein synthesis. For the gonads, the 1042 co-expressed genes enriched seminiferous tubule development. The 459 co-expressed genes identified in the liver were associated with lipid synthesis and metabolism. Finally, the cluster of 571 co-expressed genes determined in maternal blood enriched oxidative phosphorylation and thermogenesis. Next, data from the fetal organs were used to train a regression model of fetal weight, which was predicted with the maternal blood data. The best prediction was achieved when the model was trained with 35 co-expressed genes overlapping between heart and maternal blood (root-mean-square error = 0.04, R2 = 0.93). In conclusion, linking transcriptomic information from maternal blood with that from the fetal heart unveiled maternal blood as a predictor of fetal development.

Transfer of bovine embryos into a uterus primed with high progesterone concentrations positively impacts fetal development at 42 days of gestation.

Elevated circulating progesterone (P4) concentrations in the first week after conception have been associated with accelerated post-hatching conceptus elongation. However, the consequences, if any, on the development of the fetus are unknown. The objective of this study was to determine the relationship between early circulating P4 and fetal and placental morphometric characteristics at 42 days of gestation. A previously validated model of asynchronous embryo transfer (ET), known to alter uterine exposure to P4, was used in 107 heifers divided in two replicates (replicate 1: n = 51, replicate 2: n = 56). Heifers were randomly assigned to one of the two following groups: those receiving a Day 7 embryo on Day 7 of the cycle (synchronous; ET_D7, n = 49) and those transferred a Day 7 embryo on Day 9 of the cycle (asynchronous; ET_D9, n = 58). The synchronization protocol was started two days earlier for heifers in the ET_D9 group such that ET was done on the same day for both groups. P4 concentrations were determined from Day 3 after estrus to the day of ET. Pregnant heifers were slaughtered at Day 42 of gestation for fetal and placental morphometric measurements. The effects of the group, replicate, fetal sex, and interactions between these variables on fetal and placental characteristics were determined by ANOVA, while Pearson correlation was employed to assess the linear relationship between P4 concentrations two days before and on the day of ET on the fetal parameters. The uteri of heifers in the ET_D9 group were exposed to higher concentrations (P < 0.0001) of P4 from four days before ET, than heifers in the ET_D7 group. Both group and fetal sex variables impacted on fetal crown-rump length (CRL) (group: P < 0.0001, sex: P = 0.001) and fetal weight (group: P = 0.006, sex: P = 0.003). Fetal sex influenced the amniotic sac area (P = 0.003) and amniotic sac weight (P = 0.004); while the group affected the number of cotyledons (P = 0.0009), and the fetal heart weight (P = 0.018). All these parameters were larger in the ET_D9 group compared with ET_D7, and in males compared with females. There was a positive correlation between P4 concentrations two days before ET and fetal weight and CRL, for each sex or considering all fetuses (R2 ∼0.4, p < 0.05). In conclusion, bovine embryos transferred into a uterus primed with higher P4 concentrations underwent enhanced development reflected in higher weight and size at the beginning of the fetal period.

RNA-sequencing reveals genes linked with oocyte developmental potential in bovine cumulus cells.

Cumulus cells provide an interesting biological material to perform analyses to understand the molecular clues determining oocyte competence. The objective of this study was to analyze the transcriptional differences between cumulus cells from oocytes exhibiting different developmental potentials following individual in vitro embryo production by RNA-seq. Cumulus cells were allocated into three groups according to the developmental potential of the oocyte following fertilization: (1) oocytes developing to blastocysts (Bl+), (2) oocytes cleaving but arresting development before the blastocyst stage (Bl-), and (3) oocytes not cleaving (Cl-). RNAseq was performed on 4 (Cl-) or 5 samples (Bl+ and Bl-) of cumulus cells pooled from 10 cumulus-oocyte complexes per group. A total of 49, 50, and 18 differentially expressed genes (DEGs) were detected in the comparisons Bl+ versus Bl-, Bl+ versus Cl- and Bl- versus Cl-, respectively, showing a fold change greater than 1.5 at an adjusted p value <0.05. Focussing on DEGs in cumulus cells from Bl+ group, 10 DEGs were common to both comparisons (10/49 from Bl+ vs. Bl-, 10/50 from Bl+ vs. Cl-). These DEGs correspond to 6 upregulated genes (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, and SLC1A4), and 4 downregulated genes (GSTA1, PSMB8, FMOD, and SFRP4) in Bl+ compared to the other groups, from which 7 were validated by quantitative PCR (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, PSMB8 and SFRP4). These genes are involved in critical biological functions such as integrin-mediated cell adhesion, oxygen availability, IGF and Wnt signaling or PKA pathway, highlighting specific biological processes altered in incompetent in vitro maturation oocytes.

Comprehensive functional analysis reveals that acrosome integrity and viability are key variables distinguishing artificial insemination bulls of varying fertility.

In vitro methods of assessing bull semen quality in artificial insemination (AI) centers are unable to consistently detect individuals of lower fertility, and attempts to reliably predict bull fertility are still ongoing. This highlights the need to identify robust biomarkers that can be readily measured in a practical setting and used to improve current predictions of bull fertility. In this study, we comprehensively analyzed a range of functional, morphological, and intracellular attributes in cryopreserved spermatozoa from a selected cohort of Holstein Friesian AI bulls classified as having either high or low fertility (n = 10 of each fertility phenotype; difference of 11.4% in adjusted pregnancy rate between groups). Here, spermatozoa were assessed for motility and kinematic parameters, morphology, acrosome integrity, plasma membrane lipid packing, viability (or membrane integrity), superoxide production, and DNA integrity. In addition, spermatozoa were used for in vitro fertilization to evaluate their capacity for fertilization and successful embryo development. The information collected from these assessments was then used to phenotypically profile the 2 groups of bulls of divergent fertility status as well as to develop a model to predict bull fertility. According to the results, acrosome integrity and viability were the only sperm attributes that were significantly different between high- and low-fertility bulls. Interestingly, although spermatozoa from low-fertility bulls, on average, had reduced viability and acrosome integrity, this response varied considerably from bull to bull. Principal component analysis revealed a sperm phenotypic profile that represented a high proportion of ejaculates from low-fertility bulls. This was constructed based on the collective influence of several sperm attributes, including the presence of cytoplasmic droplets and superoxide production. Finally, using the combined results as a basis for modeling, we developed a linear model that was able to explain 47% of the variation in bull field fertility in addition to a logistic predictive model that had a 90% chance of distinguishing between fertility groups. Taken together, we conclude that viability and acrosome integrity could serve as fertility biomarkers in the field and, when used alongside other sperm attributes, may be useful in detecting low-fertility bulls. However, the variable nature of low-fertility bulls suggests that additional, in-depth characterization of spermatozoa at a molecular level is required to further understand the etiology of low fertility in dairy bulls.

Conceptus metabolomic profiling reveals stage-specific phenotypes leading up to pregnancy recognition in cattle†.

Reproductive efficiency in livestock is a major driver of sustainable food production. The poorly understood process of ruminant conceptus elongation (a) prerequisites maternal pregnancy recognition, (b) is essential to successful pregnancy establishment, and (c) coincides with a period of significant conceptus mortality. Conceptuses at five key developmental stages between Days 8-16 were recovered and cultured in vitro for 6 h prior to conditioned media analysis by untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. This global temporal biochemical interrogation of the ex situ bovine conceptus unearths two antithetical stage-specific metabolic phenotypes during tubular (metabolically retentive) vs. filamentous (secretory) development. Moreover, the retentive conceptus phenotype on Day 14 coincides with an established period of elevated metabolic density in the uterine fluid of heifers with high systemic progesterone-a model of accelerated conceptus elongation. These data, combined, suggest a metabolic mechanism underpinning conceptus elongation, thereby enhancing our understanding of the biochemical reciprocity of maternal-conceptus communication, prior to maternal pregnancy recognition.

JUNO protein coated beads: A potential tool to predict bovine sperm fertilizing ability.

Considerable variation in fertility exists between bulls in AI centres, despite passing minimum post-thaw quality control checks. The development of a reliable in vitro test to predict bull fertility could enable the identification and selection of high fertility bulls, without the need to resort to test inseminations. An in-depth knowledge of the molecular basis of fertilization is a prerequisite to the development of such a test or combination of tests. To date, JUNO is the only oocyte plasma membrane receptor described to be involved in gamete binding for which the partner in the sperm, IZUMO1, is known. Despite the fact that this interaction appears to be conserved among mammals, it has not been confirmed yet in some species including cattle. Furthermore, an association between binding and fertility has not been tested. Here, we propose a sperm-binding assay based on magnetic sepharose beads coated with bovine recombinant JUNO protein (BJUNO) to study sperm binding. Bull sperm bound specifically to BJUNO demonstrating that the JUNO-IZUMO1 interaction is conserved in cattle. Moreover, the assay was able to distinguish between epididymal and ejaculated sperm. Lastly, the number of sperm cells bound to BJUNO was significantly lower for frozen-thawed sperm from bulls of low vs high field fertility. In conclusion, our findings document a novel valid sperm-binding assay to predict mammalian sperm function and to investigate the role of specific proteins involved in gamete recognition and fusion.

Second-line rescue therapy with levofloxacin after H. pylori treatment failure: a Spanish multicenter study of 300 patients.

AIM: Quadruple therapy is generally recommended as second-line therapy after Helicobacter pylori (H. pylori) eradication failure. However, this regimen requires the administration of four drugs with a complex scheme, is associated with a relatively high incidence of adverse effects, and bismuth salts are not available worldwide anymore. Our aim was to evaluate the efficacy and tolerability of a triple second-line levofloxacin-based regimen in patients with H. pylori eradication failure. DESIGN: Prospective multicenter study. PATIENTS: in whom a first treatment with proton pump inhibitor-clarithromycin-amoxicillin had failed. INTERVENTION: A second eradication regimen with levofloxacin (500 mg b.i.d.), amoxicillin (1 g b.i.d.), and omeprazole (20 mg b.i.d.) was prescribed for 10 days. OUTCOME: Eradication was confirmed with (13)C-urea breath test 4-8 wk after therapy. Compliance with therapy was determined from the interview and the recovery of empty envelopes of medications. Incidence of adverse effects was evaluated by means of a specific questionnaire. RESULTS: Three hundred consecutive patients were included. Mean age was 48 yr, 47% were male, 38% had peptic ulcer, and 62% functional dyspepsia. Almost all (97%) patients took all the medications correctly. Per-protocol and intention-to-treat eradication rates were 81% (95% CI 77-86%) and 77% (73-82%). Adverse effects were reported in 22% of the patients, mainly including nausea (8%), metallic taste (5%), abdominal pain (3%), and myalgias (3%); none of them were severe. CONCLUSION: Ten-day levofloxacin-based rescue therapy constitutes an encouraging second-line strategy, representing an alternative to quadruple therapy in patients with previous proton pump inhibitor-clarithromycin-amoxicillin failure, being simple and safe.