**3. Energy sources for mammalian spermatozoa**

Energy production requires easy availability of energy substrates. In a general sense, any eu‐ karyotic cell can obtain energy from either external or internal sources. External sources can be very different, from monosaccharides to lipids, whereas internal sources are mainly poly‐ saccharides such as glycogen and lipids, although other internal sources can be aminoacids and other. Regarding mammalian sperm, separate external and also internal energy sources

have been described, offering thus a view not radically different to that observed in other eukaryotic cells. Notwithstanding, there are several characteristics that differentiates mam‐ malian sperm to other eukaryotic cells in this point. Thus, the sequence of rapid location changes that underwent spermatozoa after ejaculation implies that they are synchronically placed in very separate locations inside female genital tract. This phenomenon will imply separate availability of external energy substrates, depending on the exact placement of spermatozoa. Moreover, the majority of cells integrated in a mammalian body will obtain their external energy sources directly from blood. This is not the case of spermatozoa, which are not keeping in direct contact with blood during their entire life. In this way, external en‐ ergy sources might came from secretions of cells from the female genital tract or from semi‐ nal plasma. This implies that sperm must have very efficient mechanisms to uptake external energy resources, which will not be directly directed towards them. Moreover, and center‐ ing on seminal plasma as energy source, the time lapse that ejaculated sperm are in close contact with seminal plasma is, in fact, short. In fact, in species in which the ejaculated vol‐ ume is short and rapidly placed inside a female genital tract of large size, like cow, contact between sperm and seminal plasma is very short, indeed. In any case, after ejaculation, both sperm and seminal plasma will immediately contact with secretions from the female genital tract. In this manner, sperm would be able to simultaneously find energy sources from both seminal plasma and the female genital tract. Only in species in which the volume of the ejac‐ ulate was very voluminous, like porcine, sperm will contact seminal plasma during a signifi‐ cant time lapse. Taking into account all of this information, it seems obvious that the adequate sperm external energy sources intake will depend of two main factors. The first factor will be the efficiency of external energy sources uptake mechanisms that sperm have developed. The second factor will be the specific mixture of external energy sources that sperm find in their journey inside the female genital tract. This mixture will came from both seminal plasma and female genital tract secretions, with predominance from one or the oth‐ er source depending on the species and the exact location inside the genital tract.

the sperm motion by itself. This drop in energy requirements will be surely linked to a change in energy-fuelling pathways, although at this moment the changes suffered by ener‐

However, this is not the last change that mammalian sperm metabolism has to suffer in their life time. Once sperm reach oviduct, they rest in the oviductal crypts until their re-activa‐ tion, following ovulation. This resting step is of the utmost importance, since at this point sperm, in tightly contact with oviductal cells, reach full capacitated status [14, 55]. Capacita‐ tion implies a myriad of functional and structural changes, like loss of cell membrane cho‐ lesterol, increase in tyrosine, serine and threonine phosphorylation levels of a wide array of separate proteins and intracellular calcium mobilization, whose full description is not possi‐ ble in this chapter (see [52, 53] as reviews). Capacitation, however, has a great interest in the sense that its full achievement again implies new energy requirements to carry out processes like the increase in tyrosine phosphorylation of specific sperm proteins, such as pro-acrosin [13, 19]. This new requirements will imply again new changes in energy metabolism, which will be closely linked to the progressive changes that sperm function must suffer during this

Finally, remnant sperm will be loaded from the oviductal crypts in order to undergo oocyte penetration. In this period, capacitated sperm adopt a totally specific motility pattern known as hyperactivated motility, with separate characteristics depending on the studied species [51]. Once reached the oocyte, sperm have to penetrate it, launching a series of energy-consuming processes like adherence to oocyte zona pellucida and subsequent acrosome exocytosis [29]. Again, energy requirements will change when comparing with other sperm life–span steps. In this sense, acrosome exocytosis will need a fast and intense energy burst and, in fact, it has been described that progesterone-induced acrosome exocytosis in boar sperm that were pre‐ viously subjected to "in vitro" capacitation is simultaneous to an intense and transitory in‐ crease in O2 consumption, which would correspond to transitory mitochondria activation [43]. What are the main conclusions that can be yielded? The basic conclusion from all of this in‐ formation would be that the dramatic changes that undergo mammalian spermatozoa from ejaculation to oocyte penetration must be accompanied by concomitant, dramatic changes in their energy regulating mechanisms. Little is known regarding how mammalian sperm modulate these changes, and this is one of the most challenging investigation fields that is

Energy production requires easy availability of energy substrates. In a general sense, any eu‐ karyotic cell can obtain energy from either external or internal sources. External sources can be very different, from monosaccharides to lipids, whereas internal sources are mainly poly‐ saccharides such as glycogen and lipids, although other internal sources can be aminoacids and other. Regarding mammalian sperm, separate external and also internal energy sources

gy metabolism during this step are not well known.

156 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

currently open in the study of mammalian sperm function.

**3. Energy sources for mammalian spermatozoa**

period.

All of this digression has been only centered in the origin of sperm external energy sources. However, what are exactly these energy sources? There is a general consensus in pointing at monosaccharides as the main energy sources for mammalian sperm [46]. Notwithstanding, sperm can utilize other substances than monosaccharides. Thus, boar sperm is able to utilize a wide range of substances, such as glycerol, lactate, pyruvate and citrate [26, 27, 37]. Other species are able to utilize also non-monosaccharide substrates as energy sources, although more information is needed to clarify this point (see as examples [22, 50]). The utilization of non-monosaccharide substrates as energy sources raises the question of the usefulness of these substrates. It has been suggested that these substrates could be an alternative in cir‐ cumstances in which monosaccharide availability was limited [27, 37]. However, a thorough study of the energy substrates content that is present in each segment of the female genital tract is lacking, even in the best studied species. This impedes the complete elucidation of this suggestion. Despite this, the role of non-monosaccharide substrates as external energy sources for mammalian spermatozoa deserves a more in-depth study in order to clarify its biological importance and role.

than that of mere energy sources. In this sense, incubation of dog sperm with fructose in‐ duced a specific decrease of serine phosphorylation levels of several proteins with key roles in the regulation of sperm cell function, such as protein kinases Akt, PI3 kinase, ERK-1 and protein kinase C ([17] and Figure 1). Strikingly, the incubation with glucose induced a spe‐ cific increase in the serine phosphorylation levels of other key regulatory proteins, like c-kit, Raf-1, tyrosine kinase and several protein phosphatases ([17] and Figure 1) These effects might induce sugar-specific changes in the overall dog sperm function. On the contrary, the incubation of boar sperm with either glucose or fructose did not induce any of the specific actions observed in dog sperm ([17] and Figure 1). All of these results clearly indicate that sugars can have a sugar- and species-specific action as signaling compounds, modulating thus sperm function in a closely-linked manner, depending on the moment in which sperm and sugar were kept in contact. In this way, the idea of seminal plasma sugars as mere ener‐ gy sources would be dismissed and being substituted by another idea; sugars as both energy sources and direct sperm function modulators in a simultaneous and coordinated form.

Energy Management of Mature Mammalian Spermatozoa

http://dx.doi.org/10.5772/51711

159

External sources, however, are not the only possibility found by mammalian sperm to ob‐ tain energy. Sperm can obtain energy also from endogenous sources. One of the most stud‐ ied sources is glycogen. The presence of a functional glycogen metabolism has been demonstrated in several species, such as dog, boar, horse, ram and bonnet monkey [5], al‐ though no glycogen was found in other species like mice and rat [3]. The primary role of these internal energy sources would be, logically, the maintenance of a limited energy reser‐ voir, although this could not be a universal function. In fact, dog sperm glycogen plays a role in the achievement of "in vitro" capacitation in a medium without glucose by being a key intermediate metabolite in the obtainment of energy through gluconeogenesis, which was essential to the achievement of the capacitated status [1, 2]. Thus, in dog sperm, glyco‐ gen plays an important role as capacitation regulator, besides its energy reservoir role. In this manner, a more in-depth sturdy of the exact role of glycogen should be needed to ob‐ tain a clearer picture of the utilization of endogenous substrates as energy sources in sperm.

**4. Main metabolic pathways to obtain energy and control mechanisms involved in the coordination of energy management of mammalian**

If monosaccharides are considered as the most important exogenous energy source for mammalian sperm, the question of the precise metabolic pathways by which mature mam‐ malian sperm obtain energy is relatively straightforward. Thus, the main metabolic pathway is glycolysis. The preeminence of glycolysis in freshly ejaculated sperm has been demon‐ strated in species like bull, mice and boar [21, 33, 39]. In fact, in species like boar, at least the 95% of the energy obtained from glucose is obtained through the glycolytic pathway in freshly obtained ejaculates [33]. The explanation of the glycolysis preeminence in these spe‐ cies is easy to understand. Glycolysis would reach high velocity rates taking from a very high velocity of sugars intake and phosphorylation. This will be due to the very high sensi‐

**sperm**

**Figure 1.** Mini-array analysis of the tyrosine, serine and threonine phosphorylation status of several proteins involved in the regulation of cell cycle and overall cell function in dog and boar spermatozoa after incubation with glucose or fructose. Dog and boar spermatozoa were incubated for 5 min in the absence (C-) or presence of either 10mM fruc‐ tose (10mM F) or 10mM glucose (10mM G). The tyrosine- (Phos-Tyr), serine- (Phos-Ser) and threonine-phosphorylation (Phos-Thre) levels of each spot in the mini-arrays were then analysed. The figure shows a representative image for five separate experiments. Figure excerpted from [17]).

Turning on monosaccharides, one of the most intriguing questions is the ability of sperm to utilize a variety of sugars that are present, at east in seminal plasma. As indicated above, this is a very difficult question to study, since the sugars composition of seminal plasma is very different among species. In this sense, there are a significant number of species in which fructose is the main sugar, like human or mice [32]. However, in other species, like boar, fructose is not predominant [6, 32]. The main sugar in horse is not fructose or glucose, but sorbitol [42]. This sorbitol is further converted in fructose through the action of the en‐ zyme sorbitol dehydrogenase, despite of old works indicating that horse sperm were not able to metabolize sorbitol [42]. Finally, there are also species, like dog, which lacks any monosaccharide in its seminal plasma [44, 48]. Another intriguing question is the mere pres‐ ence of sugars, like fructose and sorbitol that are not typical in any other animal tissue. In fact, both fructose and sorbitol are typical vegetal sugars, without any significant presence in animals, excepting in mammalian seminal plasma. It is noteworthy that the utilization of sugars like fructose, sorbitol and mannose by sperm of species like boar and dog of is in fact less effective in order to obtain energy than that of glucose [36, 45]. The greater effectiveness of glucose is mainly linked to a greater sensitivity to the hexokinase system, which phos‐ phorylates sugars as a first step in their metabolization pathway [36, 45]. Taking into ac‐ count this lower efficiency, it is difficult to understand the biological logics to utilize sugars like fructose or sorbitol as mere energy sources for sperm. Another explanation for this ap‐ parent contradiction would be that non-glucose monosaccharides could exert other roles than that of mere energy sources. In this sense, incubation of dog sperm with fructose in‐ duced a specific decrease of serine phosphorylation levels of several proteins with key roles in the regulation of sperm cell function, such as protein kinases Akt, PI3 kinase, ERK-1 and protein kinase C ([17] and Figure 1). Strikingly, the incubation with glucose induced a spe‐ cific increase in the serine phosphorylation levels of other key regulatory proteins, like c-kit, Raf-1, tyrosine kinase and several protein phosphatases ([17] and Figure 1) These effects might induce sugar-specific changes in the overall dog sperm function. On the contrary, the incubation of boar sperm with either glucose or fructose did not induce any of the specific actions observed in dog sperm ([17] and Figure 1). All of these results clearly indicate that sugars can have a sugar- and species-specific action as signaling compounds, modulating thus sperm function in a closely-linked manner, depending on the moment in which sperm and sugar were kept in contact. In this way, the idea of seminal plasma sugars as mere ener‐ gy sources would be dismissed and being substituted by another idea; sugars as both energy sources and direct sperm function modulators in a simultaneous and coordinated form.

External sources, however, are not the only possibility found by mammalian sperm to ob‐ tain energy. Sperm can obtain energy also from endogenous sources. One of the most stud‐ ied sources is glycogen. The presence of a functional glycogen metabolism has been demonstrated in several species, such as dog, boar, horse, ram and bonnet monkey [5], al‐ though no glycogen was found in other species like mice and rat [3]. The primary role of these internal energy sources would be, logically, the maintenance of a limited energy reser‐ voir, although this could not be a universal function. In fact, dog sperm glycogen plays a role in the achievement of "in vitro" capacitation in a medium without glucose by being a key intermediate metabolite in the obtainment of energy through gluconeogenesis, which was essential to the achievement of the capacitated status [1, 2]. Thus, in dog sperm, glyco‐ gen plays an important role as capacitation regulator, besides its energy reservoir role. In this manner, a more in-depth sturdy of the exact role of glycogen should be needed to ob‐ tain a clearer picture of the utilization of endogenous substrates as energy sources in sperm.

**Figure 1.** Mini-array analysis of the tyrosine, serine and threonine phosphorylation status of several proteins involved in the regulation of cell cycle and overall cell function in dog and boar spermatozoa after incubation with glucose or fructose. Dog and boar spermatozoa were incubated for 5 min in the absence (C-) or presence of either 10mM fruc‐ tose (10mM F) or 10mM glucose (10mM G). The tyrosine- (Phos-Tyr), serine- (Phos-Ser) and threonine-phosphorylation (Phos-Thre) levels of each spot in the mini-arrays were then analysed. The figure shows a representative image for five

Turning on monosaccharides, one of the most intriguing questions is the ability of sperm to utilize a variety of sugars that are present, at east in seminal plasma. As indicated above, this is a very difficult question to study, since the sugars composition of seminal plasma is very different among species. In this sense, there are a significant number of species in which fructose is the main sugar, like human or mice [32]. However, in other species, like boar, fructose is not predominant [6, 32]. The main sugar in horse is not fructose or glucose, but sorbitol [42]. This sorbitol is further converted in fructose through the action of the en‐ zyme sorbitol dehydrogenase, despite of old works indicating that horse sperm were not able to metabolize sorbitol [42]. Finally, there are also species, like dog, which lacks any monosaccharide in its seminal plasma [44, 48]. Another intriguing question is the mere pres‐ ence of sugars, like fructose and sorbitol that are not typical in any other animal tissue. In fact, both fructose and sorbitol are typical vegetal sugars, without any significant presence in animals, excepting in mammalian seminal plasma. It is noteworthy that the utilization of sugars like fructose, sorbitol and mannose by sperm of species like boar and dog of is in fact less effective in order to obtain energy than that of glucose [36, 45]. The greater effectiveness of glucose is mainly linked to a greater sensitivity to the hexokinase system, which phos‐ phorylates sugars as a first step in their metabolization pathway [36, 45]. Taking into ac‐ count this lower efficiency, it is difficult to understand the biological logics to utilize sugars like fructose or sorbitol as mere energy sources for sperm. Another explanation for this ap‐ parent contradiction would be that non-glucose monosaccharides could exert other roles

separate experiments. Figure excerpted from [17]).

158 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed
