Effects of a walnut-enriched diet on murine sperm and epididymis: its impact on plasma membrane calcium ATPase-4 (Pmca4) null mice
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Male factors account for 50% of infertile couples, with 25% of these cases being idiopathic. Sperm defects are the single most common cause of male infertility and it is believed that genetic factors play a major role in idiopathic cases. Recently, lifestyle choices have also been shown to impact sperm function, both positively and negatively. Positive effects have resulted from a diet enriched in walnuts, which are high in polyunsaturated fatty acids (PUFAs) and antioxidants: when this diet was administered to men on a Western diet for 12 weeks there was a significant increase in sperm motility, vitality, and normal morphology. To date, a study of the effects of a walnut-enriched diet on sperm quality has not been conducted in an animal model and was one of the goals of the present study. Here it is shown that a walnut-enriched diet (accounting for 19.6% caloric intake) given to male mice for 9-11 weeks, significantly improved sperm motility and morphology. Thus, the beneficial effects of a walnut enriched diet on sperm quality were confirmed in the murine model.
To determine if this diet could have a beneficial effect on sperm with a motility defect due to a genetic mutation, Pmca4 null male mice, in which the Plasma Membrane Calcium ATPase 4 (Pmca4) Ca2+ efflux pump is missing, were subjected to the diet. Deletion of Pmca4, the major Ca2+ efflux mechanism in murine sperm, results in infertility due to loss of hyperactivated motility by a mechanism that remains unclear. To explore the possible underlying mechanism, various properties of Pmca4 null sperm were examined and attempts were made to modulate them by exposure to the diet. Although motility rates were not affected by the administration of the diet in Pmca4 nulls, the potential impacts of Pmca4 deletion on membrane integrity were determined by studying three sperm characteristics (vitality, morphology, and acrosomal reaction rates). Membrane integrity, as assessed by vitality staining, and the ability to acrosome react showed no significant difference between control and KO or between diet and no-diet. On the other hand, morphologically normal sperm were significantly decreased in the Pmca4 KO control, compared to control WT; and in diet-treated animals normal morphology was significantly increased for both WT and KO sperm. The levels of lipid peroxidation showed a trending increase in Pmca4 null, compared to WT control sperm, and significantly decreased in both WT and KO sperm following administration of the diet. Thus lipid peroxidation was determined to be a potential mechanism resulting in the loss of motility and the increase in morphologically abnormal sperm, the majority of which were tail defects, in Pmca4 nulls.
Testicular germ cells were also affected by Pmca4 deletion, as shown by the TUNEL assay, for which there was a significant increase in spermatogonia (p<0.005) and spermatocytes (p<0.05) in nulls, compared to WT males. This was confirmed by hematoxylin and eosin (H&E) staining, which revealed increased disorganization of the seminiferous tubule structure. Pmca4 KO epididymides were analyzed via H&E staining and revealed cellular damage associated with apoptosis. There was a significant increase in vacuolization of epithelial cells in the cauda in the Pmca4 KO males, as well as an increased number of pyknotic-rounded cells. These results support a mechanism in which Pmca4 deletion results in loss of motility, increased tail defects, and apoptosis via oxidative stress and lipid peroxidation. These findings give support for the addition of dietary supplements for the maintenance of reproductive health, as a potential lifestyle modification to increase reproductive success.