80 following letter code: each age group was assigned a letter (3m=a, 6m=b, 9m=c, 12m=d, 24m=e). Letters above the bars denote significance between respective groups. Upper-case letters indicate statistical significance at the 0.05 level and lower-case significance at the 0.001 level.
81 male reproductive system by a direct antioxidant activity [29]. There was a striking decrease in testes creatine content in older rats, suggesting that despite sperm continues to be produced, the conditions for the development of suitable male germ cells are altered. In fact, decreased creatine content was observed in testicular tissue after exposure to ethylene glycol monoethyl ether and was associated with deficient primary and pachytene spermatocytes [30].
Thus, the decreased creatine content can be associated with deficient germ cells development, which is also corroborated by the increased availability of lactate in older rats [31]. We also evaluated the protein expression of LDH and MCT4, which is responsible for the export of lactate from Sertoli cells, where it is produced, to developing germ cells. The observed decrease in the amount of MCT4 in the testes of older rats (6 to 12 months) corroborates the possible lactate accumulation in Sertoli cells and decreased bioavailability to the developing germ cells. Sharp increase in MCT4, presumably of compensatory nature, was observed in the oldest group of animals. Although several aging related histological and functional studies have been done in rats, they differ significantly in the choice of aims and age time-points.
Furthermore, differences have been observed between various rat breeds [32]. However, the results of our study seem to complement the studies performed on Wistar rats using the same or similar time-points. After the initial rise from 3 months-of-age, the peak of capillary density, as well as in the number of spermatocytes I and late spermatides was observed in animals of 6 to 9 months-of age [33]. These parameters undergo sharp decline at 12 months with the concomitant increase in degenerated seminiferous tubules. Also, at the age of 6 months, a decrease of the Leydig cells [34] and the first changes in the levels of genes and enzymes involved in steroidogenesis were observed [35], suggesting the beginning of compromised spermatogenesis.
The aging related changes in testes have also been associated with increased oxidative stress and decline in antioxidant defences, including superoxide dismutase, glutathione peroxidase and glutathione reductase activities [36]. Our results support this observation since we observed a decrease in most antioxidant metabolites like betaine, creatine, taurine and GSH oxidation product GSSG. However, recovery in testicular taurine levels observed at 9 and 12 months may be a compensatory mechanism for the observed loss of antioxidant activity and elevated ROS levels caused by aging. In agreement with our findings, taurine is reported as the major free β-amino acid in the male reproductive system [37]. It acts as a capacitating agent [38] and sperm motility factor [39]. Moreover, the significant elevation of taurine precursor hypotaurine at the age of 12 and 24 months could present an additional strategy to counterbalance oxidative stress, since it is an excellent in vivo radical scavenger [40]. The loss
82 of antioxtidative capacity was supported by the observed increase in oxidative damage, particularly in proteins.
In the presence of high metabolic rates, as those noted in spermatogenesis, mitochondrial OXPHOS may induce ROS overproduction [41]. Frequently, mitochondrial complex I significantly contributes to the generation of free radicals [42, 43]. This trend seems associated with the bioenergetics profile of the testicular tissue, rather than the whole metabolic profile, although these are intimately associated. In testes of older mice (up to 24 months-of-age), we observed increased levels of complex I protein which might be linked with increased ROS production. Still, this increase was also accompanied by an increase in the expression levels of other OXPHOS complexes. This increased expression of mitochondrial complexes with age may serve as a compensatory mechanism to counteract the loss of mitochondrial function that is associated with aging. However, in the older animals (24 months-of-age rats), the expression levels of OXPHOS complexes decreased significantly (when compared to 12 months-of-age rats), which suggests the impairment of mitochondrial function, as noted by others [44].
Testicular lipid metabolism, in particular phospholipid metabolism is significantly altered by aging. In testes, there is a high number of proliferating germ cells that require constant lipid membrane synthesis and a large pull of substrates. Phosphocholine is a major precursor in phospholipid membrane synthesis and could serve as a specific marker for spermatogenesis [45]. Ethanolamine is another phospholipid precursor. Some of the key enzymes in phospholipid biosynthesis, like choline kinase or phosphatidylserine synthase isoform PSS2, are highly expressed in testes illustrating the relevance of this process [46]. The levels of both choline and ethanolamine increased in the testes with advanced age (12 and 24 months-of- age) and were accompanied by decreasing levels of their phosphorylated products, suggesting lower rates of phospholipid synthesis and thus compromised spermatogenesis.
Concentrations of other phospholipid precursors, myo-inositol and glycerol, also increased in the testis of the same age groups. Phospholipid metabolism is essential for normal reproductive health and the deletion of genes responsible for the expression of phosphocholine cytidylyltrasnferase isoform CTβ2 and phosphatidylserine synthase isoform PSS2 lead to testicular dystrophy and dysfunction [46]. At the same time, increased levels of glycerophosphocholine suggested increased phospholipid degradation. Choline is a precursor in the synthesis of betaine, another metabolite with antioxidant properties. However, unlike choline, betaine levels in testes decrease with age, starting at 12 months-of-age. Although the exact mechanism of its antioxidative activity remains unknown, it was suggested to be through modulation of methionine-homocystein cycle, by increasing the concentrations of S- adenosylmethionine and methionine [47]. Betaine participates in the cycle by donating methyl
83 groups necessary for conversion of S-adenosylhomocysteine to S-adenosylmethionine.
Dimethylglycine, which results from such methyl exchange, follows betaine decrease trend.
The described changes illustrate perturbations in the cellular methylation potential, which is necessary for various biosynthetic and regulatory reactions, since S-adenosylmethionine serves as universal methyl donor and participates in methylation of DNA, proteins and in the synthesis of small molecules like creatine, carnitine and phosphatidylcholine. Previous studies described a gradual loss of general DNA methylation with aging in humans influencing epigenetic patterns [48] and link it to age-related diseases [49].
Carnitine transports fatty acids from cytoplasm to mitochondria where they are oxidized and form acetyl coenzyme A, which can then enter TCA to be further oxidized. Alternatively, it can be transported back into cytosol and used in fatty acid or sterol synthesis. General loss in carnitine with aging has been reported [50] but in testes its levels increase with age. That, however, was not accompanied by a concomitant increase in TCA cycle activity, suggesting other players in the process, namely the reduced levels of succinate that indicate reduction of TCA cycle activity and intermediate pools. However, so far it is not clear whether the onset of reduced TCA activity from 6 months-of-age on is the result of changes in anaplerotic or cataplerotic contributions to the cycle. Recently, a comparison of age-related gene expression between different animal models, C. elegans and skin isolated from zebrafish and mouse, led to the discovery that the catabolism of BCAAs is consistently regulated in all three organisms [51] and that BCAA diet supplementation improved both life and healthspan in C. elegans.
Age-related changes in BCAA metabolism were also found in murine muscle and liver [52] and were linked with mitochondrial biogenesis dysfunction [53] and oxidative stress reduction [54].
The increase in BCAAs observed in our model further supports the importance of BCCAs in organ specific response to age-related changes. Apart from BCAAs, significant changes were observed in metabolism related to phenylalanine, tyrosine and glutamate. Although selected blood plasma amino acids were singled out as potential predictive markers of old age in mouse [55], their biological significance and relevance for testis and reproductive processes has yet to be elucidated.
Nucleic acid metabolism is also affected by aging. The metabolic profiles relate intricate interplay between different energetic and biosynthetic needs of a highly metabolically active organ that involve this class of compounds in an age dependent manner. Although changes in relative concentrations might reflect different contributions of metabolic pathways, including biosynthesis, degradation and salvaging, it seems that young age of 3 months might be dominated by increased nucleotide synthesis (IMP, CMP, ATP), which might be linked to mitochondrial health and activity.
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