Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs

Abstract

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the 'Entrepelado' and 'Lampiñ o' breeds. The samples were suspended in a commercial extender and refrigerated to 17 8C for transport to the laboratory (step A), where they were further extended with a lactose-egg yolk-based extender and chilled to 5 8C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS-PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the 'Entrepelado' breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing-thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.

Investigation of relationship between expression of the glucose transporter3 (GLUT3) and sperm quality in Asian elephants (Elephas maximus)

“…These GLUT3 proteins were localized at the principal and end piece of the sperm tail, with the exclusion of the middle piece. However, the localization was different from the sperm of other mammals; for example, in boar, positive GLUT3 was evident in the acrosome and in a band across the middle of the sperm head (5,6,19,20). In bull sperm, the positive GLUT3 signal was present only in the middle piece (8,19).…”

Spermiomics: A New Term Describing the Global Survey of the Overall Sperm Function by the Combined Utilization of Immunocytochemistry, Metabolomics, Proteomics and Other Classical Analytical Techniques

“…The most common of the techniques utilized to determine both the total content and phosphorylation levels of a specific protein is the Western blotting of transferred samples after being subjected to an electrophoresis onto an SDS-polyacrylamide support (SDS-PAGE). Again, this chapter is not devoted to the description of this technique, although it is necessary to indicate that Western blotting is currently one of the most widely utilized techniques in the study of mammalian sperm function, existing a very high amount of articles published in which this technique has been utilized to analyze proteins like hexose transporters (Rigau et al, 2002;Sancho et al, 2007), protein kinases (Vijayaraghavan et al, 1997;Breitbart & Naor, 1999) or even nucleoproteins (Flores et al, 2008;Flores et al, 2011). The usefulness of Western blotting is evident, although it is a technique that can cause many troubles to the unwary.…”

“…In this way, troubles related with the specific fixative technique utilized can be of importance. For example, fixation of samples with ethanol rendered a different location of several hexose transporters in boar sperm when compared with similar samples fixed with paraformaldehyde (Rigau et al, 2002;Sancho et al, 2007;Bucci et al, 2010). In this way, there are several applications of immunocytochemistry that are completely valid for spermatozoa.…”

Study on Changes of Sperm Head Morphometry and Dna Integrity of Freeze-Dried Bovine Spermatozoa

“…In cattle and other mammalian have been widely studied the effects of cryopreservation on sperm function and fertility. The reduction of sperm quality in cryopreserved semen and reveals that temperatures under 5°C seem to be a major factor in explaining the failure of sperm to survive the freezing-thawing process (Sancho et al, 2007). Various sperm organelles have been known to be affected due to the detrimental effects of cryopreservation.…”