Sperm miR-15a and miR-29b are associated with bull fertility.

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting. MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility. MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility.

Sperm cellular and nuclear dynamics associated with bull fertility.

The objective of this study was to ascertain cellular characteristics and the dynamics of the sperm chromatin proteins protamine 1 (PRM1) and protamine 2 (PRM2) in the sperm of Holstein bulls having a different fertility status. Important sperm variables were analyzed using computer-assisted sperm analysis (CASA). Sperm membrane, acrosome status, DNA integrity were also assessed using propidium iodide (PI), fluorescein isothiocyanate conjugated to Arachis hypogaea (FITC-PNA), and acridine orange (AO) followed by flow cytometry. In addition, abundances of PRM1 and PRM2 were analyzed using flow cytometry experiments. Differences in sperm decondensation capacity were assessed in bulls of varying fertility using a decondensation assay. As determined using CASA, average pathway velocity, amplitude of lateral head displacement and straightness were different (P < 0.05) for sperm from high and low fertility bulls. There, however, were no differences between the high and low fertility bulls for characteristics of sperm plasma membrane, acrosome, and DNA integrity (P > 0.05). Relative abundances of PRM1 and PRM2 in sperm from the high and low fertility bulls were inversely related (P < 0.0001). Percentages of decondensed sperm were different between high and low fertility bulls (P < 0.0001) and total numbers of decondensed sperm were greater in low fertility bulls than high fertility bulls (R2 = 0.72). Results of the present study are significant because molecular and morphological phenotypes of sperm that were detected affect fertility in livestock species.

Expression dynamics of Integrin Subunit Beta 5 in bovine gametes and embryos imply functions in male fertility and early embryonic development.

Integrins have been shown to act as signalling receptors, and they primarily recognise extracellular matrix ligands on the oocyte surface. However, their possible roles in oocyte activation and embryo development are not clearly understood. The objectives of this study were to evaluate expression of Integrin Subunit Beta 5 (ITGß5) in bovine sperm, oocytes, and early embryos and to ascertain the evolutionary conservation of ITGß5. To accomplish these objectives, we used western blotting to study expression levels of ITGß5 protein in sperm and RT-qPCR to determine expression levels of ITGß5 transcripts in oocytes and embryos. We have also used bioinformatic analysis to determine the evolutionary conservation of the ITGß5 protein among various species. Western blotting showed that ITGß5 protein was detectable in bull sperm. Moreover, results of RT-qPCR showed that levels of ITGß5 were significantly higher in the two-cell embryos, followed by the 8-16-cell embryos. However, no significant difference in expression levels were noted for the morula and blastocyst stages as compared to MII oocytes. Bioinformatic analysis revealed that ITGß5 is conserved among various species. We conclude that expression of ITGß5 in bovine gametes and embryos implies an important role in fertilisation and embryogenesis.

Testis specific histone 2B is associated with sperm chromatin dynamics and bull fertility-a pilot study.

BACKGROUND: Bull fertility is the degree of sperm's ability to fertilize and activate the egg and support embryo development, and this is critical for herd reproductive performance. We used the bull as a unique model organism for the study of male fertility because cattle genetics and physiology is similar to those of other mammals including humans. Moreover, reliable fertility data along with well-established in vitro systems are available for bovine. The objective of this original study was to ascertain evolutionary diversification and expression dynamics of Testis Specific Histone 2B (TH2B) in sperm from Holstein bulls with different fertility scores. METHODS: The intensity of TH2B was determined by using flow cytometry in sperm from 13 high and 13 low fertility bulls. Expression levels of TH2B were measured using immunofluorescence and Western blotting in sperm from five high and five low fertility bulls. Sequence identity, evolutionary distance and interactome of TH2B were evaluated by dotmatcher, STRING and Cytoscape. Data were analyzed using linear mixed effects model and regression plots were drawn. RESULTS: The intensity of TH2B as measured by flow cytometry was significantly affected by an interaction between fertility group and fertility score (P = 0.0182). The intensity of TH2B in sperm from the high fertility group decreased (P = 0.0055) as fertility increased. TH2B was constantly detectable in sperm and expression levels of TH2B decreased in relation to fertility in sperm from the high fertility group (P = 0.018). TH2B biological functions include male gamete generation, chromosome organization, DNA packaging, DNA conformation change, chromatin organization, nucleosome organization, chromatin disassembly, spermatid nucleus elongation, spermatid nucleus differentiation, sperm motility, chromatin organization, chromatin condensation, chromatin silencing, nucleus organization, and chromatin remodeling (P < 0.05). CONCLUSIONS: We elucidated the cellular localization and molecular physiology of TH2B using both computational and cell biology approaches. In addition to advancing the fundamental science of mammalian male gamete, the present findings can be potentially used to evaluate semen quality and predict male fertility in the future. TRIAL REGISTRATION: This study did not involve any live animals. We did not perform any anesthesia, euthanasia, or any kind of animal sacrifice. The cryopreserved semen samples were obtained from Alta Genetics, Inc., Watertown, WI, USA. All samples were preserved in liquid nitrogen.

Further evidences for the mode of action of the larvicidal m-pentadecadienyl-phenol isolated from Myracrodruon urundeuva seeds against Aedes aegypti.

Nowadays, dengue fever is considered the most important arbovirosis worldwide and its control is still based upon combating the vector Aedes aegypti. Besides monitoring of mosquito populations resistant to conventional insecticides, the search for new environmentally safe insecticides and conduction of molecular studies focusing on the elucidation of mode of action and possible resistance mechanisms are considered the key for a sustainable management of the mosquito vector. Thus, the present work aimed to assess changes in protein expression of 3rd-instar larvae of Ae. aegypti after exposure to the natural insecticide m-pentadecadienyl-phenol. Bidimensional electrophoresis followed by mass spectrometry resulted in identification of 12 proteins differentially expressed between control and treated groups. Larvae exposed to the toxic compound for 24h showed elevated detoxification response (glutathione-S-transferase), increased levels of stress-related proteins (HSP70) as well as evidence of lysosome stabilization to enable survival. Furthermore, expression of proteins involved in protection of peritrophic membrane and metabolism of lipids indicated systemic effect of toxic effects in treated larvae.