Pregestational fructose-induced metabolic syndrome in Wistar rats causes sexually dimorphic behavioral changes in their offspring.

Metabolic syndrome (MetS), marked by enduring metabolic inflammation, has detrimental effects on cognitive performance and brain structure, influencing behavior. This study aimed to investigate whether maternal MetS could negatively impact the neurodevelopment and metabolism of offspring. To test this hypothesis, 2 months old female Wistar rats were subjected to a 10-week regimen of tap water alone or supplemented with 20% fructose to induce MetS. Dams were mated with healthy males to generate litters: OC (offspring from control dams) and OMetS (offspring from dams with MetS). To isolate prenatal effects, all pups were breastfed by control nurse dams, maintaining a standard diet and water ad libitum until weaning. Behavioral assessments were conducted between postnatal days (PN) 22 and 95, and metabolic parameters were analyzed post-sacrifice on PN100. Results from the elevated plus maze, the open field, and the marble burying tests revealed a heightened anxiety-like phenotype in OMetS females. The novel object recognition test showed that exclusively OMetS males had long-term memory impairment. In the reciprocal social interaction test, OMetS displayed a lower number of social interactions, with a notable increase in "socially inactive" behavior observed exclusively in females. Additionally, in the three-chamber test, social preference and social novelty indexes were found to be lower solely among OMetS females. An increase in visceral fat concomitantly with hypertriglyceridemia was the relevant postmortem metabolic finding in OMetS females. In summary, maternal MetS leads to enduring damage and adverse effects on offspring neurobehavior and metabolism, with notable sexual dimorphism.

Lipid peroxidation and neuroinflammation: A possible link between maternal fructose intake and delay of acquisition of neonatal reflexes in Wistar female rats.

Fructose is a common sweetener found in the daily diet supplemented to many processed and ultra-processed foods and beverages. Consumption of fructose-sweetened beverages has drastically increased in the last decades and is widely associated with metabolic disease, systemic pro-inflammatory status, and adverse transgenerational effects. To date, the impact of maternal fructose intake in brain function of the offspring is less explored. Therefore, the aim of this study was first, to investigate adverse effects in developmental milestones of the progeny of mothers with metabolic syndrome (MetS), induced by ad libitum consumption of a 20% fructose solution, and second to identify possible molecular changes in the nervous system of the newborns associated with maternal fructose intake. Wistar rats were randomly separated into two groups with access to water or fructose (20% w/v in water) for 10 weeks. After MetS was confirmed, dams were mated with control males and continued drinking water or fructose solution during gestation. At postnatal day (PN) 1, a subgroup of offspring of each sex was sacrificed and brains were dissected for oxidative stress and inflammatory status analysis. Changes in the developmental milestones due to maternal fructose consumption were studied (PN3-PN21) in another subgroup of offspring. Sexually dimorphic effects were found on the progeny's acquisition of neurodevelopmental milestones, in brain lipid peroxidation, neuroinflammation, and antioxidative defensive response. Our results suggest that dams' MetS, induced by fructose intake, disrupts brain redox homeostasis in female offspring and affects sensorimotor brain circuitry which may have a translational value for studying neurodevelopmental diseases.

Elovl4 and Fa2h expression during rat spermatogenesis: a link to the very-long-chain PUFAs typical of germ cell sphingolipids.

The sphingolipids (SLs) of rodent spermatogenic cells (spermatocytes, spermatids) and spermatozoa contain nonhydroxylated and 2-hydroxylated versions of very-long-chain (C26-C32) PUFAs (n-V and h-V, respectively) not present in Sertoli cells (SCs). Here, we investigated the expression of selected fatty acid elongases [elongation of very-long-chain fatty acid protein (Elovl)], with a focus on Elovl4, and a fatty acid 2-hydroxylase (Fa2h) in rat testes with postnatal development and germ cell differentiation. Along with Elovl5 and Elovl2, Elovl4 was actively transcribed in the adult testis. Elovl4 mRNA levels were high in immature testes and SCs, though the protein was absent. The Elovl4 protein was a germ cell product. All cells under study elongated [3H]arachidonate to tetraenoic and pentaenoic C24 PUFA, but only germ cells produced C26-C32 PUFAs. Spermatocytes displayed the highest Elovl4 protein levels and enzymatic activity. Fa2h mRNA was produced exclusively in germ cells, mostly round spermatids. As a protein, Fa2h was mainly concentrated in late spermatids, in the step of spermiogenesis in which they elongate and their heads change shape. The expression of Elovl4 and Fa2h thus correlate with the abundance of n-Vs and h-Vs in the SLs of rat spermatocytes and spermatids, respectively.

The E2F2 transcription factor sustains hepatic glycerophospholipid homeostasis in mice.

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2+/+) and E2F2 deficient (E2F2-/-) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2+/+ and E2F2-/- mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2-/- mice resembles the phenotype of proliferating E2F2+/+ liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.

Altered lipid homeostasis in Sertoli cells stressed by mild hyperthermia.

Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC). Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days) of rat testes to 43 °C led to accumulation of neutral lipids. This SC-specific lipid function took 1-2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43 °C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (α-tubulin) and microfilament (f-actin) organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster.

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs.

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21-22°C), the rest remaining in the liquid state in the 5°C-45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.

Uneven distribution of ceramides, sphingomyelins and glycerophospholipids between heads and tails of rat spermatozoa.

Previous work showed that rat germ cells and spermatozoa contain ceramides and sphingomyelins with high proportions of nonhydroxy and 2-hydroxy (2-OH) polyunsaturated fatty acids (PUFA) with very long chains (VLCPUFA). The aim of this study was to assess how these lipids are distributed between the heads and tails of mature spermatozoa in comparison with other membrane lipid classes. In addition to quantitative differences due to the fact that these gametes have a long, voluminous tail and a minute head, several compositional dissimilarities emerged between these two regions. The total cholesterol/total phospholipid ratio, the choline/ethanolamine glycerophospholipid (ChoGpl/EtnGpl) ratio, and the proportion of plasmalogens within these two classes, were much larger in the head than in the tail. Whereas EtnGpl was rich in 22:5n-6 in both regions, ChoGpl had plenty of 22:4n-9, especially in the heads. An important proportion of the head EtnGpl- 22:5n-6 and ChoGpl 22:4n-9 was in plasmenyl- (rather than in phosphatidyl-) subclasses. The heads concentrated all of the sphingomyelin species with nonhydroxy- and 2-OH VLCPUFA, and the tails most of the saturated fatty acids that are present in total sperm sphingomyelin. Unexpectedly, virtually all of the abundant spermatozoal ceramides, predominantly made up by species with 2-OH VLCPUFA, was located in the tail. The fact that intact rat spermatozoa constitutively have much more VLCPUFA-containing ceramide than sphingomyelin is explained by the present findings, since the former are mostly lipids of the large tail while the latter mostly collect in the small head.

Mild testicular hyperthermia transiently increases lipid droplet accumulation and modifies sphingolipid and glycerophospholipid acyl chains in the rat testis.

Spermatogenesis is known to be vulnerable to temperature. The aim of this study was to investigate the effects on testicular lipids of the transient germ cell loss that is induced by mild testicular hyperthermia. Adult rat testes were exposed once a day to 43 °C for 15 min for 5 days and the effects were followed for several weeks. Two week after the last heat exposure, spermatocytes and early spermatids had virtually disappeared and the seminiferous tubules were populated mostly by mature spermatids and spermatozoa. One week later, the latter were also absent and mostly Sertoli cells populated the tubules. During these 3 weeks, glycerophospholipids (Gpl) and triacylglycerols with long-chain polyunsaturated fatty acids (PUFA) (e.g., 22:5n-6) and species of sphingomyelin and ceramide with nonhydroxy and 2-hydroxy very long-chain (VLC) PUFA (e.g., 28:4n-6, 2-OH 30:5n-6) decreased alongside the germ cells. Concomitantly, the amounts of cholesteryl esters and ether-linked triglycerides increased, both lipids accumulating long-chain and very-long-chain polyenes. This concurred with a considerable buildup of lipid droplets in Sertoli cells, evidently containing these neutral lipids, apparently formed during germ cell-derived membrane lipid catabolism. Between week 4 and week 6, new cohorts of spermatocytes appeared, and by week 12 most cell changes were reversed. Accordingly, as germ cell differentiation proceeded, 22:5n-6-rich Gpl augmented and spermatocyte-associated sphingolipids with nonhydroxy VLCPUFA appeared before their 2-hydroxy counterparts. The unique fatty acids of rat testicular lipids after mild hyperthermia reveal lipid catabolic and biosynthetic reactions that occur in normal spermatogenesis.

Differentiation-related changes in lipid classes with long-chain and very long-chain polyenoic fatty acids in rat spermatogenic cells.

In rat seminiferous tubules (ST), cells that contain polar and neutral lipids with long-chain polyenoic fatty acids (PUFA) and sphingomyelins (SM) and ceramides (Cer) with very long chain (VLC) PUFA of the n-6 series coexist. In this study, pachytene spermatocytes and round spermatids were isolated to determine how these lipids change during spermatogenesis. As the amount per cell of PUFA-rich glycerophospholipids (GPL) decreased with cell size, the 22:5/20:4 ratio increased with cell differentiation. The elovl2 and elovl5 genes, required for 22:5 formation, were expressed (mRNA) in both cell types. Residual bodies- particles with compacted organelles and materials discarded from late spermatids-concentrated cholesterol, 22:5-rich triacylglycerols, and GPL, including plasmalogens and phosphatidylserine. Species of SM and Cer with nonhydroxylated (n-) VLCPUFA (28:4, 30:5, and 32:5) predominated in pachytene spermatocytes, whereas species with the corresponding 2-hydroxy (2-OH) VLCPUFA prevailed in round spermatids. Thus, a dramatic increase in the 2-OH/n-VLCPUFA ratio in SM and Cer was a hallmark of differentiation. A substantial decrease of 2-OH SM occurred between spermatids and mature spermatozoa and 2-OH SM species were collected in residual bodies "en route" to Sertoli cells. Notably, spermatids and spermatozoa gained a significant amount of ceramides devoid of n-VLCPUFA but having 2-OH VLCPUFA as their main fatty acids.

Changes in lipids containing long- and very long-chain polyunsaturated fatty acids in cryptorchid rat testes.

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C(18)-C(22)) and very long-chain (VLC) (C(24)-C(32)) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7-10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.

Very long-chain polyunsaturated fatty acids are the major acyl groups of sphingomyelins and ceramides in the head of mammalian spermatozoa.

Very long-chain (C24 to C34) polyunsaturated fatty acids (VLCPUFA) are important constituents of sphingomyelin (SM) and ceramide (Cer) in testicular germ cells. In the present paper we focused on the SM and Cer and their fatty acids in spermatozoa and their main regions, heads and tails. In bull and ram spermatozoa, SM was the third most abundant phospholipid and VLCPUFA were the major acyl groups ( approximately 70%) of SM and Cer. In rat epididymal spermatozoa the SM/Cer ratio was low in the absence of and could be maintained high in the presence of the cation chelator EDTA, added to the medium used for sperm isolation. This fact points to the occurrence of an active divalent cation-dependent sphingomyelinase. Bull and rat sperm had an uneven head-tail distribution of phospholipid, with virtually all the VLCPUFA-rich SM located at the head, the lower SM content in the rat being determined by the lower sperm head/tail size ratio. Most of the SM from bull sperm heads was readily solubilized with 1% Triton X-100 at 4 degrees C. The detergent-soluble SM fraction was richer in VLCPUFA than the nonsoluble fraction and richer in saturated fatty acids. Cer was produced at the expense of SM, thus decreasing severalfold the SM/Cer ratio in rat spermatozoa incubated for 2 h in presence of the sperm-capacitating agents, calcium, bicarbonate, and albumin. The generation of Cer from SM in the sperm head surface may be an early step among the biochemical and biophysical changes known to take place in the spermatozoon in the physiological events preceding fertilization.

Ceramides and sphingomyelins with high proportions of very long-chain polyunsaturated fatty acids in mammalian germ cells.

Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.

Very long chain PUFA in murine testicular triglycerides and cholesterol esters.

Very long chain (VLC) PUFA of the n-6 and n-3 series are known to occur in mammalian testis. The aim of this work was to characterize further two testicular lipid classes with VLCPUFA, cholesterol esters (CE) and total triglycerides (TG) in rat and mouse testis. The VLCPUFA predominating in these lipids were a series of n-6 pentaenes and tetraenes with 24 to 32 carbons, including small amounts of odd-chain PUFA, 28:5n-6 and 24:5n-6 prevailing in CE and TG, respectively. Most of the VLCPUFA of TG were concentrated in a small fraction of TG, made up by 1-O-alkyl-2,3-DAG. This TG subclass was absent altogether from the TG of sexually immature testis. The TG and the CE with VLCPUFA only occurred in testis of adult fertile animals. The proportion of VLCPUFA in total TG and CE was higher in rodents than in other mammals. In the n-6 PUFA-rich adult mouse testis, the amounts of testicular triacylglycerols decreased significantly after consumption of fish oil for 2 wk. Whereas 18:2n-6 was significantly reduced, the amounts of 22:5n-6 and longer n-6 PUFA were less affected in all major testicular lipids including PC and PE, where they were unchanged. The 1-O-alkyl-2,3-DAG and their n-6 VLCPUFA were virtually unaffected by the diet. The VLCPUFA-containing molecular species of CE and TG may represent a form of storage of cholesterol and polyenoic FA required to sustain spermatogenesis. Via chain-shortening, VLCPUFA stored in the neutral lipids may serve as precursors of the major C22 PUFA typical of cell membrane glycerophospholipids, protecting testicular cells against shifts in FA composition induced by dietary changes.

Reversibility of the changes induced by n-3 fatty acids in mouse plasma, liver and blood cell lipids.

The changes induced by dietary n-3 fatty acids (FA) in the lipids and FA of plasma, liver and blood cells, and their reversibility, was studied in mice given a diet containing 9% fish oil (FO) for 2 weeks and then returned to, and kept for another 2 weeks on, the usual standard lab chow diet. In plasma, the concentrations of phospholipids (PL), mostly phosphatidylcholine (PC), triacylglycerols (TG), cholesterol and cholesterol esters (CE) decreased rapidly after starting the FO diet, and remained low from day 3 onwards. This decrease was concomitant with a remarkable reduction in the n-6 FA, especially 18:2n-6, not compensated for by the relative enrichment in n-3 FA induced by FO. In liver, TG and CE decreased and PL slightly increased, all of them showing reduced n-6/n-3 ratios. Sphingomyelin, which lacks polyunsaturated FA other than small amounts of 18:2 and 24:2n-6, showed altered ratios between its very long chain monoenes and saturates. In the washout phase, the most rapid event was an immediate increase in 18:2n-6 and after a few days in 20:4n-6 in plasma and liver, where most of the lipid and FA changes were reversed completely in about 10 days. In the case of blood cells even 2 weeks were insufficient for a reversal to the initial n-6/n-3 ratios. The lipid class responsible for this lack of reversibility was phosphatidylethanolamine, PC having returned to the initial fatty acid composition during the stated period.