Molecular and Cellular Correlates of Sperm Viability Associated with Male Fertility

Fertility is classified as one of the most limiting aspects plaguing successful mammalian reproduction. In addition, sequential collaborations between a quality spermatozoon and oocyte are essential for successful fertilization, and key in subsequent embryonic development. Male fertility has been consistently declining in mammals. Currently, cost efficient and reliable methods predicting fertility are lacking. As a result, the objectives of this research were to establish molecular markers to predict male fertility by determining sperm cellular phenotype variability among bulls of varying fertility by focusing on the roles of protein markers, TUBB 2C, HSP10, HXK1 and SOD1; establish expression characteristics of ITGB5 protein in sperm, oocytes and early embryos; and finally; determine expression level variability and functions of miRNAs, miR-214 and miR-25 in sperm from bulls of varying fertility. We found that while no significant differences occurred in the expression levels of our proteins in high and low fertility bulls, correlations were discovered between SOD1, HSPE1 and fertility, and subpopulations consisting of intact, partially damaged and completely damaged acrosomes in flow cytometry and microscopy experiments were identified. Results also showed that expression levels of itgb5 were significantly higher in the 2-cell bovine 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 the MII oocytes. Phylogenetic analyses confirmed conservation of itgb5 across multiple species, further indicating its potential importance and functional role(s) in fertilization and potential as a marker of fertility. Micro-RNA studies revealed differential expression levels of miR-25 and miR- 214 for all samples analyzed, yet there was no significant difference in expression of the microRNAs when comparing high fertility and low fertility bulls. Also, processes associated with spermatogenesis, such as cellular growth, transduction, translation and transformation which are necessary for fertility, were shown to be targets of miR-214 and miR-25. These results imply that sperm proteins and sperm-bore miRNAs could potentially be targeted as molecular markers of fertility. The findings are important because they illuminate the molecular and cellular underpinnings of gamete quality that influence successful fertilization and early development

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 (ITGβ5) in bovine sperm, oocytes, and early embryos and to ascertain the evolutionary conservation of ITGβ5. To accomplish these objectives, we used western blotting to study expression levels of ITGβ5 protein in sperm and RT-qPCR to determine expression levels of ITGβ5 transcripts in oocytes and embryos. We have also used bioinformatic analysis to determine the evolutionary conservation of the ITGβ5 protein among various species. Western blotting showed that ITGβ5 protein was detectable in bull sperm. Moreover, results of RT-qPCR showed that levels of ITGβ5 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 ITGβ5 is conserved among various species. We conclude that expression of ITGβ5 in bovine gametes and embryos implies an important role in fertilisation and embryogenesis

Sperm superoxide dismutase is associated with bull fertility

Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of β-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (P\u3c0.05) and that SOD1 is the best protein to diagnose bulls based on the fertility index (P\u3c0.05). Using Metacore analysis, we identified an SOD1 network with pathways and linkages with other relevant molecules. We concluded that SOD1 sperm expression is associated with in vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility

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 superoxide dismutase is associated with bull fertility

Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of β-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (P\u3c0.05) and that SOD1 is the best protein to diagnose bulls based on the fertility index (P\u3c0.05). Using Metacore analysis, we identified an SOD1 network with pathways and linkages with other relevant molecules. We concluded that SOD1 sperm expression is associated with in vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility