PUFA-rich phospholipid classes and subclasses of ram spermatozoa are unevenly affected by cryopreservation with a soybean lecithin-based extender.

Cryopreservation is known to affect spermatozoa structure and function. Ram sperm are among the most highly sensitive mammalian gametes to freezing, due to their lipid composition, which limit their efficiency in artificial insemination programs. The aim of this study was to investigate the effects of cryopreservation with a chemically defined soybean lecithin-based extender on ram spermatozoa functionality on the one hand, and quantifiable changes in lipid and fatty acid profile on the other. Freeze-thawing decreased sperm quality, as indicated by post-thaw parameters related to membrane integrity, mitochondrial viability and sperm motility. The most relevant lipid change after cryopreservation was a remarkable loss of all glycerophospholipids containing 22:6n-3. Species of sphingomyelin with very long chain polyunsaturated fatty acids (VLC-PUFA), that are exclusively located in the sperm head, where responsible of its reduction after cryostorage. Freezing caused a reduction in mitochondrial function, which was confirmed by significantly decreased of mitochondrial membrane potential and by the generation of 4-HNE. Mitochondria damage was accompanied by a loss in cardiolipin with 18:2n-6 and phosphatidylethanolamine with 20:4n-6, two well-known lipids that are critical components for mitochondrial membrane functionality. Loss of sterols after cryopreservation occurred along with a decrease in the order of sperm membrane lipids. Our research provides new insights on deleterious effects of cryopreservation on PUFA-rich phospholipids of ram sperm and highlight their importance as biomarkers of ultrastructural, biochemical and functional damage that ram spermatozoa undergo after freezing-thawing.

The inhibition of microtubule dynamics instability alters lipid homeostasis in TM4 Sertoli cells.

Sertoli cells (SC) structurally support and transport nutrients to germ cells during spermatogenesis facilitated by an active cytoskeleton. Chemical perturbation of SC microtubule (MT) dynamics instability leads to premature germ cell exfoliation demonstrating that this process is essential for male fertility, yet the effects of MT damaging drugs on SC lipid metabolism have been less explored. The aim of this study was to advance our understanding of how adequate SC MT dynamicity is needed to finely tune lipid homeostasis. To elucidate the role of MT dynamics instability on the latter, we suppressed MT dynamicity by long-term exposures to 10 nM of nocodazole (NCZ) on TM4-SC cultures. Inhibition of MT dynamics instability affected the distribution of [3H] arachidonate on TM4-SC. Triacylglycerols (TAG) exhibited a higher proportion of the [3H] label, with significantly lower percentages in the mitochondrial phospholipid cardiolipin, and notably, also in phosphatidylethanolamine. A noteworthy and progressive accumulation of lipid droplets during the period of exposure to NCZ was accompanied by increased TAG levels but not cholesterol levels in TM4-SC. NCZ-exposed cells reduced their mitochondrial membrane potential and increased ROS production without triggering apoptosis, had a compromised autophagic flux, and lost their transferrin expression. Although SC morphology was preserved, the NCZ-exposed cells displayed alteration of the normal organization of microfilaments (f-actin) and intermediate filaments (vimentin). Our findings suggest that a preserved MT dynamicity is essential in the maintenance of lipid and fatty acids homeostasis in SC, and thus highlights a novel target in these cells for drugs that impair MT dynamicity.