Uncovering sperm metabolome to discover biomarkers for bull fertility.

BACKGROUND: Subfertility decreases the efficiency of the cattle industry because artificial insemination employs spermatozoa from a single bull to inseminate thousands of cows. Variation in bull fertility has been demonstrated even among those animals exhibiting normal sperm numbers, motility, and morphology. Despite advances in research, molecular and cellular mechanisms underlying the causes of low fertility in some bulls have not been fully elucidated. In this study, we investigated the metabolic profile of bull spermatozoa using non-targeted metabolomics. Statistical analysis and bioinformatic tools were employed to evaluate the metabolic profiles high and low fertility groups. Metabolic pathways associated with the sperm metabolome were also reported. RESULTS: A total of 22 distinct metabolites were detected in spermatozoa from bulls with high fertility (HF) or low fertility (LF) phenotype. The major metabolite classes of bovine sperm were organic acids/derivatives and fatty acids/conjugates. We demonstrated that the abundance ratios of five sperm metabolites were statistically different between HF and LF groups including gamma-aminobutyric acid (GABA), carbamate, benzoic acid, lactic acid, and palmitic acid. Metabolites with different abundances in HF and LF bulls had also VIP scores of greater than 1.5 and AUC- ROC curves of more than 80%. In addition, four metabolic pathways associated with differential metabolites namely alanine, aspartate and glutamate metabolism, ß-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism were also explored. CONCLUSIONS: This is the first study aimed at ascertaining the metabolome of spermatozoa from bulls with different fertility phenotype using gas chromatography-mass spectrometry. We identified five metabolites in the two groups of sires and such molecules can be used, in the future, as key indicators of bull fertility.

Acetylation and methylation of sperm histone 3 lysine 27 (H3K27ac and H3K27me3) are associated with bull fertility.

Epigenetic modifications in histones are crucial for proper sperm physiology, egg activation and reproductive development of males. The objectives of this study were to determine the conservation and interactomes of histone three (H3) and ascertain the expression dynamics of acetylated and methylated H3 lysine 27 (H3K27ac and H3K27me3) in spermatozoa from Holstein bulls with different fertility. Methods in immunocytochemistry and flow cytometry were used to evaluate the expression dynamics of H3K27ac and H3K27me3 in spermatozoa from 10 bulls with different in vivo fertility. Computational biology methods including Clustal Omega and Cytoscape were performed to determine the evolutionary conservation and interactome of H3. The post-translational modifications (PTM) of H3 (H3K27ac and H3K27me3) had different spatiotemporal dynamics in the sperm head. Intensities of methylation were higher than those of acetylation and inversely correlated between the two fertility groups (p = .0032). The interacting proteins of H3 are involved in critical subcellular processes such as regulation of methylation, nucleosome assembly, regulation of DNA replication and chromatin assembly. These results are significant because they help advance fundamental science and biotechnology of mammalian reproduction.

Transcriptome analysis of bull spermatozoa: implications for male fertility.

Spermatozoa deliver more than the paternal genome into the oocyte; they also carry remnant messenger RNA from spermatogenesis. The RNA profiles of spermatozoa from high-fertility and a low-fertility Holstein bulls were analysed using Affymetrix bovine genechips. A total of 415 transcripts out of approximately 24,000 were differentially detected in spermatozoa collected from both bulls (fold change > or =2.0; P<0.01). These transcripts were associated with different cellular functions and biological processes. Spermatozoa from high-fertility bulls contained higher concentrations of transcripts for membrane and extracellular space protein locations, while spermatozoa from the low-fertility bulls were deficient of transcripts for transcriptional and translational factors. Quantitative real-time PCR was used on three low-fertility and four high-fertility bulls to validate the microarray data. Two highly represented transcripts in the microarray analysis (protamine 1 and casein beta 2) were validated, as well as a third transcript (thrombospondin receptor CD36 molecule) that showed a lower concentration in low-fertility bulls. This study presents the global analysis of spermatozoa originating from bulls with opposite fertility. These results provide some specific transcripts in spermatozoa that could be associated with bull fertility.

Proteomic landscape of seminal plasma associated with dairy bull fertility.

Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho = 0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.

Genetics of insulin dependent diabetes mellitus in Israel: population and family study.

The association between insulin dependent diabetes mellitus (IDDM) and the HLA system was studied in two groups of Jewish patients: 50 Ashkenazim and 42 non-Ashkenazim. The pattern of association of HLA-A and B locus antigens was somewhat different from that observed in European Caucasian patients. HLA-B8 had a higher frequency; B15 and Cw3 were rare in the population studied and were less frequent in IDDM patients than in controls. On the other hand, the frequency of A26, B18, and Bw38 was increased in Ashkenazi patients, but not in non-Ashkenazim, who in turn showed an increase for Bw51. Although the association between IDDM and HLA-A and B locus antigens shows a marked variability in different populations, the association with HLA-DR3 and DR4 is constant feature. There was a typical excess of DR3/DR4 heterozygotes in both patient groups. This heterozygote type carries the highest relative risk, followed by DR4/DR4 homozygotes. These data can well be interpreted by a model of two different HLA-linked susceptibility genes, one associated with DR3 and the other one with DR4, that interact so that different genotypes are associated with different levels of penetrance. This model received further support from studies in 15 multiple case families where there is an excess of affected sib pairs sharing two DR antigens.

Dietary adjustment during self-blood-glucose monitoring in pregnant women with insulin-dependent diabetes mellitus.

The interaction between the daily distribution of carbohydrates and frequent self-blood-glucose monitoring (SBGM) was studied in 13 pregnant women who had had diabetes for 4 to 19 years. Before and during SBGM, data were obtained on dietary history, daily blood glucose levels, and HbA1C. Optimal control was found with 3 main meals and 5 snacks. The total daily caloric intake decreased without change in the proportions of protein, fat, and carbohydrate. Consumption of starch increased, and that of simple sugars decreased. Although no changes were made in the daily amount of insulin, the women's diabetic control improved significantly.

Induction of tissue eosinophilia by platelet-activating factor in Merino sheep.

A study was undertaken on the capacity of platelet-activating factor (PAF) to induce eosinophil accumulation in the mammary glands of non-lactating sheep. Platelet-activating factor induced dose-dependent accumulation of eosinophils in mammary exudates 24 h after infusion. Infection, by intraruminal injection of 20,000 infective Trichostrongylus colubriformis larvae, did not modify the responsiveness of outbred sheep to intramammary infusion of PAF. Mature ewes from high and low responder lines of a flock of sheep, selected on the basis of their responses to vaccination and experimental challenge with T. colubriformis as lambs, did not differ in the magnitude of the eosinophil responses to doses of PAF from 5 x 10(-13) to 5 x 10(-7) mol per gland. Intramammary infusion of an extract from third stage larvae of Haemonchus contortus elicited inflammatory exudates containing five- to ten-fold more eosinophils than that elicited by the highest dose of PAF tested. The experiments indicate that the eosinophil chemotactic agonist PAF can induce tissue eosinophilia in sheep and thus may play a role in directing the accumulation of eosinophils in tissues during disease states such as gastrointestinal parasitism.

The effect of systemic treatment with platelet-activating factor on the migration of eosinophils to lung, pleural and peritoneal cavities in the guinea pig.

Subcutaneous treatment of guinea pigs with platelet-activating factor (PAF) caused an increase in the prevalence of eosinophils in lavage fluid recovered from pulmonary airways, and pleural and peritoneal cavities. In PAF-treated animals, total numbers of eosinophils and macrophages in washings were positively correlated, thus there was no apparent competition between the two cell types for migration at traffic sites into body cavities. The results indicate that PAF acts centrally to enhance the migration of eosinophils and monocytes into body cavities, perhaps by inducing bone marrow release of both classes of leucocytes which may then migrate constitutively into lung, pleural and peritoneal cavities.

Infantile methemoglobinemia caused by food additives.

A small outbreak of toxic methemoglobinemia occurred among infants in a pediatric ward. Investigation revealed that the most likely source of toxicity was an approved fat preservative which had been added to a soybean infant formula by the manufacturer. This fat preservative contained three phenolic compounds having highly effective antioxygenic properties (butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate). The outbreak ceased when the offending agents were eliminated from the food preparation. It is emphasized that the approval of chemicals for use in the food industry through toxicity studies does not necessarily guarantee against the hazards of toxicity, especially during infancy.

Folate nutrition and growth in infancy.

The effect of suboptimal folate nutrtion on the growing infant was studied in a population of infants fed a diet based on boiled, pasteurised cows' milk. One group of infants received a daily supplement of 1 mg folic acid from age 2 months, while the other group received a placebo. The infants were seen at bimonthly intervals. In the supplemented group the red cell folate level had increased to twice its pretreatment value by 4 months, and remained at this high level to the end of the first year. Hb concentration and incidence of anaemia were similar in both groups. The incidence of infection in the two groups did not differ. Weights and lengths attained at 6 months, and the rate of gain from 2 to 6 months were higher in infants whose folate levels were above the median value than in those below it. In the second half of the first year the differences between the two groups were no longer evident.

Oral clonidine as a growth hormone stimulation test.

A single oral dose of 0.15 mg/m2 of clonidine was given to eighteen healthy children and adolescents and to seven patients with hypopituitarism. In healthy subjects there was a pronounced increase in plasma growth hormone from 4.9 +/- 1.3 ng/ml (+/- SEM) to 34.4 +/- 4.5 ng/ml. In the patients with hypopituitarism there was no change in growth-hormone concentrations. Clonidine induced a slight increase in blood glucose in healthy subjects and a slight decrease in patients with hypopituitarism. During the test systolic blood-pressure decreased by a mean of 20 mm Hg in the healthy subjects and by 25 mm Hg in the patients with hypopituitarism. The only side-effect observed was drowsiness. Oral administration of clonidine, a well-tolerated drug, seems to be a simple test for of GH reserves in children and adolescents.

Radiographic appearance of nutrient canals in the region of the erupting permanent maxillary cuspid.

A retrospective radiographic search was made for nutrient canals situated immediately inferior to the incisal tip of the unerupted permanent maxillary cuspid. In this study, the structure was observed radiographically in 35.4% of the children between the ages of 6 and 10 years, with an apparently greater incidence among girls.

Pyogenic granuloma as a cause of bone loss in a twelve-year-old child: report of case.

The pyogenic granuloma frequently appears between the ages of eleven and forty years. It is a benign soft tissue lesion of inflammatory origin, which may be misdiagnosed as a neoplastic tumor due to its rapid development. Definitive diagnosis can only be made by histopathologic examination. Treatment is by conservative local excision. The present manuscript reports on the diagnosis and treatment of such a lesion, which caused significant maxillary bone loss in a twelve-year-old child.

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.

Comparison of the effect of insulin hypoglycemia and clonidine on secretion of growth hormone, cortisol and beta-endorphin in children and adolescents.

Plasma beta-endorphin, human growth hormone (hGH) and cortisol were measured concomitantly during insulin hypoglycemia (0.1 u/kg i.v.) or clonidine administration (0.075 mg/m2 orally) in children with idiopathic short stature. Whereas hypoglycemia raised plasma beta-endorphin levels, clonidine slightly decreased beta-endorphin in six subjects and had no effect in four. Cortisol levels increased following hypoglycemia and decreased markedly after clonidine. hGH increased to greater than 20 ng/ml in all but one subject. The findings are interpreted as further evidence that the hGH stimulation of clonidine is not stress-mediated.

Plasma growth hormone response to oral clonidine as compared to insulin hypoglycemia in obese children and adolescents.

The response of plasma growth hormone (hGH) to a single oral dose of clonidine (0.15 mg/m2) was compared with that obtained with insulin hypoglycemia (ITT) induced by administration of double the usual dose (0.2 U/kg i.v.) in 13 obese subjects aged 5-17 years (7 males, 6 females) with a subscapular skinfold greater than 20 mm and a weight greater than 2 SD of the median. Six healthy subjects (3 males, 3 females), aged 8-14 years who served as controls received the usual dose of 0.1 U/kg i.v. in the ITT. Clonidine induced an increase of more than 10 ng/ml in the plasma hGH levels in 10 (4 males, 6 females) of the 13 obese subjects and in all of the healthy controls, with peak levels ranging from 14.3 to 31.0 ng/ml (m +/- SD 21.0 +/- 5.2 ng/ml); the ITT elicited a similar rise in only 6 of the 13 subjects and 3 of the healthy controls, with peak levels ranging from 9.8 to 20.0 ng/ml (m +/- SD 14.4 +/- 4.5 ng/ml). Clonidine decreased plasma insulin levels in all the obese female subjects (by a mean of 65%) whereas in the obese males the insulin pattern was variable. There was no change in blood glucose levels following the administration of clonidine; during the ITT all subjects showed a decrease to less than 50 mg/dl. Blood pressure decreased by a mean of 20 mmHg during the clonidine test. This study demonstrates that clonidine is a more effective hGH stimulus than insulin induced hypoglycemia in normal and in obese children and that the lower hGH secretion of the obese is confirmed by the clonidine test.