Aging is a natural inevitable process that affects all organisms and leads a series of physiological changes in our bodies. One of the changes related with aging is decreased
Chapter 5
Concluding Remarks
117 fertility, a matter assigned greater clinical relevance, and commonly neglect how aging affects male fertility.
Infertility has a broad effect on public health. In developed countries, where expectancy of a prolonged life is well-known, modern trends have imposed the delay of first parenthood [2].
Aging is explained by biological and demographic topics characterized
by the impairment of several physiological functions and to what concern human reproduction, age related decrease of couples fertility potential is usually associated to female aging [2, 3].
This has guided many to believe that aging has insignificant effects on male reproductive capacity, and that men would therefore have an almost endless reproductive life. However, several studies establish a direct association between aging and structural and functional changes of sexual organs and the endocrine system, that in turn suggest an effect on sperm parameters and fertility [1, 4].
In most cases, studies are concentrated on the clinical aspect of reproductive aging and insufficient possible molecular mechanisms responsible for the decline in male fertility have been unveiled. In addition, studies in humans rely on sperm parameters, which are limited forecasters of male fertility, and external insults (e.g. smoking, alcohol, environmental contaminants) limit the interpretation and make conclusions difficult. Thus,in addition to human studies, animal studies are also extremely important to unravel the mechanisms by which aging modifies male reproductive function. In this work, we used the NMR-based metabolomics to identify metabolic changes associated with different stages of reproductive maturity, from the onset to senescence.
Seminal plasma as has huge potential as a clinical sample for non-invasive diagnostics and has been used as an important source for investigating male infertility [5]. Hamamah et al.
conducted one of the first studies that used seminal plasma for the identification of biomarker using NMR technology [6]. The seminal plasma proteome contains a large amount of tissue- specific proteins that might precisely designate a pathological process in the tissue of origin.
Moreover, it also has an enormous amount of potential protein biomarkers, much more abundant than those found in blood serum or urine, and, therefore, are more easily identified and quantified in semen by mass spectrometry and other techniques [5].
Several of the constituents found in seminal plasma are well described, and few have proven clinical relevance [7-9]. In recent years, it has become progressively more evident that it is not only the sperm quality that is required for a successful pregnancy but that it is also of extreme importance the composition of seminal fluid [10, 11].
Metabolomics is a recently developed technique for the analysis of the small-molecule component of biological systems and samples [12]. Is a rapid and non-invasive analysis, that
118 the functional phenotype in a cell, tissue or organism [13]. Metabolites consist of a very varied group of organic small molecules, comprising organic acids, amino acids, sugars, phosphosugars, steroids, and biogenic amines [12].
Important modifications have been observed, among semen samples of men with spermatogenesis failure, obstructive azoospermia, oligoasthenoteratozoospermia and healthy donors in the content of various metabolites (such us citrate, lactate, glycerylphosphorylcholine and glycerylphosphorylethanolamine). Furthermore, it was shown that levels of certain of oxidative stress biomarkers (-CH, -NH, -OH and ROH) demonstrated on the seminal plasma of healthy men were consistently different from those of patients with idiopathic infertility, varicocele and vasectomy reversal [13]. In our work, several testicular metabolic pathways were affected as well as themetabolism of human seminal fluid.
In our experiments, numerous testicular metabolic pathways were affected as early as 6 months-of-age. Testicular cells, and particularly developing germ cells, require large extracellular concentrations of creatine to maintain cell viability and function [14]. Indeed, creatine signals are the most predominant in NMR spectra of rat testis up to 9 months-of-age.
Creatine serves as energy buffer and helps replenishing ATP levels in tissues with high-energy demand, though its exact role in spermatogenesis remains unknown. In addition, it might exert activity in the male reproductive system by a direct antioxidant activity [15]. 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. There was also a significant decrease in seminal fluid creatine content in G2, suggesting that the antioxidant activity is altered in male in this age group.
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 [16]. In rats, 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. MCT4 increase, 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 [17]. 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
119 concomitant increase in degenerated seminiferous tubules. Also, at the age of 6 months, a decrease of the Leydig cells [19] and the first changes in the levels of genes and enzymes involved in steroidogenesis were observed [20], suggesting the beginning of compromised spermatogenesis.
The increased oxidative stress and decline in antioxidant defences, including superoxide dismutase, glutathione peroxidase and glutathione reductase activities have been associated with aging in testes [21]. Our results support this observation since we observed in rat testes 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 old rats 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 [22]. It acts as a capacitating agent [23] and sperm motility factor [24]. Moreover, the significant raise 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 [25]. The loss of antioxidative 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 [26]. Commonly, mitochondrial complex I significantly contributes to the generation of free radicals [27, 28]. This tendency 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. 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 [29].
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 [30]. Some of the key enzymes in phospholipid biosynthesis, like choline kinase or
120 relevance of this process [31]. 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 [31]. 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.
Betaine participates in the cycle by donating methyl groups necessary for conversion of S- adenosylhomocysteine to S-adenosylmethionine. The described changes illustrate perturbations in the cellular methylation potential, 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. Former studies described a gradual loss of general DNA methylation with aging in humans influencing epigenetic patterns [32] and link it to age- related diseases [33].
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. Loss in carnitine with aging has been descrived [34] 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. Age-related changes in BCAA metabolism were found in various organisms namely C. elegans and skin isolated from zebrafish and mouse [35] murine muscle and liver [36] and were linked with mitochondrial biogenesis dysfunction [37] and oxidative stress reduction [38]. The increase in BCAAs observed in our model further supports the importance of BCCAs in organ specific response to age-related changes. Significant changes were also observed in metabolism related to phenylalanine, tyrosine and glutamate.
In our experiments using human seminal fluid, we continue to observe an increase with age in BCAAs (isoleucine, leucine and valine), when analysing all the samples together, and the significant increase of Leucine in AZ patients.
Amino acids isoleucine and leucine are responsible for delaying calcium uptake by ejaculated sperm by altering active calcium transport across the sperm plasma membrane [39]. The presence of these two metabolites in seminal plasma regulates the fertility potential of sperm
121 reaction regulating their function [40].
Phenylalanine, tyrosine, and histidine or their derivatives are precursors of catecholamines or neurotransmitters [41, 42]. Numerous studies suggest that the increase in sexual interest may be linked with elevated levels of the brain neurotransmitter, dopamine. In our study, we observed a decrease with age in the amounts of phenylalanine, tyrosine, and histidine, which, although not statistically significant, is very evident. However, when analysing the metabolites according to the different age groups used in this work, the decrease of tyrosine with age becomes significant in the younger group G1. This seems to support the fact that with increasing age the sexual interest tends to diminish.
Also in G1, a significant decrease in glutamine (GLN) was observed. GLN is an essential amino acid, which plays a central role in the response to injury and also preserves the stores of antioxidants in tissues since it is a precursor of glutathione [43]. In our experiments involving human seminal fluid samples, a significant decrease in GLN was observed. Thus, this decrease in GLN may indicate that there is a greater possibility of damage occurring to important cellular components caused by reactive oxygen species with age in this specific group.
It is well known that glutamate has an important role in controlling gonadotropin-releasing hormone (GnRH) neuron excitability. The GnRH neuronal network is the leading controler of the reproductive axis [44]. In our work was observed an increase for glutamate in G2 and NZ patients. This may indicate that there is an impairment in the activity of the GnRH neurons with increasing age.
Compared with other body fluids, seminal plasma contains higher concentrations of uridine, and it is known that concentrations are increased significantly in vasectomized men compared with normal men [45]. Human seminal plasma contain very high concentrations of uridine.Low concentrations of uridine in prostatic secretions and very high concentrations of uridine in seminal plasma compared with other body fluids were
found in patients with prostatitis. This suggests a role for uridine in the aetiology of prostatitis [46]. An important function of uridine could be to promote sperm motility, as seminal plasma uridine concentrations are positively correlated to percentage sperm motility [46, 47]. In our work G2 group presents a significant decrease with age in the amounts of Uridine. Therefore, this decrease seems to indicate the existence of an impairment in the beginning and success of the reproduction process with advancing age.
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
122 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.
The aging related changes in semen samples have been associated with reduction in sperm parameters [48]. Some studies established that there is a decreasing trend of pregnancy after intercourse with men older than 34 years. This occurs, regardless of the woman's age, and that this trend increases with age [49] .There are several studies where male age is associated with a decrease in semen volume, decrease in total sperm count, decrease in motility, decrease in progressive motility, decrease in percent normal sperm and an increase in DNA fragmentation. However, male age appeared to have no consistent effect on sperm concentration. That is why it is important to point out that total sperm count derives from sperm concentration and semen volume. Thus, the observed decline in total sperm count with age is driven mainly by the decline in semen volume with age, rather than a decline in sperm concentration with age [4].
In our study, we observed a significant increase in sperm concentration with age, which corroborates previous studies in which it was observed that the sperm concentration at age ≥ 55 was 19.8% higher compared with value at age ≥ 45 to < 55, and 34.8% higher when compared with the values from patients < 25 years old [48]. Furthermore, we observe an inverse relationship between semen volume and patient age. After 40 years of age semen volume decreases, although this value is not statistically significant, it represents an evident decrease. This result is in agreement with that observed in another study in which, in addition to observe a significant decrease in seminal volume and sperm quality with increasing age, they also concluded that the most significant reduction in sperm parameters occurs after 55 years of age [48].The phenotypes of physiological and pathological testicular aging have been well studied; however, the causes, mechanisms and related rescue measures remain largely unknown [50].
To conclude, as far as we know this constitutes the first metabolomics study in the field of testes aging. Overall results describe the application and the potential of NMR-based metabolomics in the study of age-related changes in metabolic pathways that lead to testicular senescence, and in seminal plasma metabolites quantification. We identified the metabolites associated with each stage of reproductive maturity from the very onset to the senescence in rats and 37 metabolites in human seminal fluid of patient undergoing ART. Although at the present moment no threshold can be established, the decrease in the testicular content of most antioxidant metabolites, the changes in phospholipids synthesis and the decrease in nucleotide synthesis from 3 months-of-age onwards illustrate a clear decline in testicular
123 process, and to correlate the observed changes in testis metabolome with its functionality as well as with the normal sperm maturation and function.
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