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

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‐ tivity to sugars of both GLUTs monosaccharide transporters and overall hexokinase activity. In fact, glycolytic rate is so high in species like bull that it rarely achieve the theoretical stoi‐ chiometric ATP yield of the glycolytic pathway, living thus to the establishment of an active substrate cycling, important to the maintenance of motility [21]. In fact, the ability of gener‐ ate energy of a specific sugar would depend on the ability of each sugar to be uptaken and subsequent phosphorylated. In this way, it is important to remind that this sensitivity changes upon a sugar- and species-specific basis. Thus, as described above and taking por‐ cine sperm as a basis, it is important to remind that the velocity by which glucose is phos‐ phorylated and then incorporated to the glycolytic flux is greater than that observed by other sugars like fructose, sorbitol and mannose [36]. The ability of each species to utilize each separate sugar will be then different, depending on the specific machinery that sperm have in order to uptake and further phosphorylate monosaccharides. In fact, this machinery can be more different among species than that previously thought. As an example, dog sperm have two separate hexokinase activities. The first has a very high sensitivity for sug‐ ars as glucose, with a Km of about 0.1 mM. The second hexokinase activity has much lover glucose sensitivity, with kinetic properties very similar to those described for hepatic gluco‐ kinase [16]. This sophisticated machinery makes dog sperm able to develop a dual reactivity to react against very separate glucose concentrations, specifically changing sperm function in contact with environments with these separate characteristics. Remarkably, sperm from other species such as boar have not any glucokinase-like activity [16], reflecting thus a spe‐ cies-specific reactivity against glucose that is inititated at the very start of the glucose utiliza‐ tion pathway.

sults seem to indicate that the minority mitochondrial respiration is essential to obtain a fea‐

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**Figure 2.** Rhythm of O2 consumption of boar sperm subjected to "in vitro" capacitation and subsequent "in vitro" ac‐ rosome reaction in the presence or absence of olygomycin A or in Ca2+-depleted capacitation medium. Boar sperm were incubated for 4h and then were added with 10 µg/mL progesterone and subjected to a further incubation for 60 min. A): Sperm cells incubated in a standard capacitation medium or in media added with 2.4 µM olygomycin A. : Control cells. : Spermatozoa incubated in capacitation medium added with 2.4 µM olygomycin A from the begin‐ ning of the incubation. ▲: Spermatozoa incubated in a standard capacitation medium for 4h and subsequent added with 10 µg/mL progesterone and 2.4 µM olygomycin A together. B): Sperm cells incubated in a standard capacitation medium or in Ca2+-depleted media. ○: Spermatozoa incubated in capacitation medium without Ca2+ and added with 2 mM EGTA from the beginning of the experiments. : Spermatozoa incubated in a standard capacitation medium for 4h and subsequent added with 10 µg/mL progesterone and 2 mM EGTA together Results are expressed as means ±S.E.M. for 7 separate experiments. Asterisks indicate significant (P<0.05) differences when compared with the respec‐

However, as exposed above, monosaccharides are not the only energy source that sperm can utilize. Other substrates, such as citrate and lactate, can be utilized to obtain energy, at least in several mammalian species. The ways by which mammalian sperm utilize these non-

tive Control values. Results excerpted from [43]) and unpublished data from our laboratory.

sible progesterone-induced acrosome exocytosis.

The final sugar utilization step is the entry of pyruvate obtained at the end of glycolysis into mitochondria to be subsequent degraded into the mitochondrial respiration system. There is not a universal agreement regarding the importance of mitochondria-based energy obtain‐ ment. In this way, an optimal mitochondrial function has been related not only with sperm motility in bull [18], horse [20], ram [34] and mouse [39] but also with fertilization ability in human (30). However, gene knock-out of the glycolytic enzyme glyceraldehyde-phosphate dehydrogenase (GAPDH) in transgenic mice caused the appearance of non-motile sperm and a significant reduction of the ATP content (10% of the total) despite having no deficien‐ cy in oxygen consumption [38]. This seems to imply that although a correct mitochondrial function is needed to the maintenance of an optimal sperm function, mitochondrial respira‐ tion would not be the most important role of mitochondria to exert their activity. Another explanation would be that mitochondrial respiration would not be important in the mainte‐ nance of the overall energy status of sperm, although it would be of the utmost importance in the maintenance of punctual aspects of sperm function. In this sense, progesterone-in‐ duced acrosome exocytosis of boar sperm subjected to a previous "in vitro" capacitation is concomitant with a rapid, intense and transitory burst of oxygen consumption [42]. More‐ over, unpublished results from our laboratory show that the inhibition of this oxygen con‐ sumption burst is concomitant with an almost complete lack of progesterone-induced acrosome exocytosis (Figure 2 and data not shown). These results are concomitant with overall low levels of oxygen consumption, which in fact indicate that the majority of ATPs obtained by boar sperm do not come from mitochondrial respiration [33]. However, the re‐ sults seem to indicate that the minority mitochondrial respiration is essential to obtain a fea‐ sible progesterone-induced acrosome exocytosis.

tivity to sugars of both GLUTs monosaccharide transporters and overall hexokinase activity. In fact, glycolytic rate is so high in species like bull that it rarely achieve the theoretical stoi‐ chiometric ATP yield of the glycolytic pathway, living thus to the establishment of an active substrate cycling, important to the maintenance of motility [21]. In fact, the ability of gener‐ ate energy of a specific sugar would depend on the ability of each sugar to be uptaken and subsequent phosphorylated. In this way, it is important to remind that this sensitivity changes upon a sugar- and species-specific basis. Thus, as described above and taking por‐ cine sperm as a basis, it is important to remind that the velocity by which glucose is phos‐ phorylated and then incorporated to the glycolytic flux is greater than that observed by other sugars like fructose, sorbitol and mannose [36]. The ability of each species to utilize each separate sugar will be then different, depending on the specific machinery that sperm have in order to uptake and further phosphorylate monosaccharides. In fact, this machinery can be more different among species than that previously thought. As an example, dog sperm have two separate hexokinase activities. The first has a very high sensitivity for sug‐ ars as glucose, with a Km of about 0.1 mM. The second hexokinase activity has much lover glucose sensitivity, with kinetic properties very similar to those described for hepatic gluco‐ kinase [16]. This sophisticated machinery makes dog sperm able to develop a dual reactivity to react against very separate glucose concentrations, specifically changing sperm function in contact with environments with these separate characteristics. Remarkably, sperm from other species such as boar have not any glucokinase-like activity [16], reflecting thus a spe‐ cies-specific reactivity against glucose that is inititated at the very start of the glucose utiliza‐

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

The final sugar utilization step is the entry of pyruvate obtained at the end of glycolysis into mitochondria to be subsequent degraded into the mitochondrial respiration system. There is not a universal agreement regarding the importance of mitochondria-based energy obtain‐ ment. In this way, an optimal mitochondrial function has been related not only with sperm motility in bull [18], horse [20], ram [34] and mouse [39] but also with fertilization ability in human (30). However, gene knock-out of the glycolytic enzyme glyceraldehyde-phosphate dehydrogenase (GAPDH) in transgenic mice caused the appearance of non-motile sperm and a significant reduction of the ATP content (10% of the total) despite having no deficien‐ cy in oxygen consumption [38]. This seems to imply that although a correct mitochondrial function is needed to the maintenance of an optimal sperm function, mitochondrial respira‐ tion would not be the most important role of mitochondria to exert their activity. Another explanation would be that mitochondrial respiration would not be important in the mainte‐ nance of the overall energy status of sperm, although it would be of the utmost importance in the maintenance of punctual aspects of sperm function. In this sense, progesterone-in‐ duced acrosome exocytosis of boar sperm subjected to a previous "in vitro" capacitation is concomitant with a rapid, intense and transitory burst of oxygen consumption [42]. More‐ over, unpublished results from our laboratory show that the inhibition of this oxygen con‐ sumption burst is concomitant with an almost complete lack of progesterone-induced acrosome exocytosis (Figure 2 and data not shown). These results are concomitant with overall low levels of oxygen consumption, which in fact indicate that the majority of ATPs obtained by boar sperm do not come from mitochondrial respiration [33]. However, the re‐

tion pathway.

**Figure 2.** Rhythm of O2 consumption of boar sperm subjected to "in vitro" capacitation and subsequent "in vitro" ac‐ rosome reaction in the presence or absence of olygomycin A or in Ca2+-depleted capacitation medium. Boar sperm were incubated for 4h and then were added with 10 µg/mL progesterone and subjected to a further incubation for 60 min. A): Sperm cells incubated in a standard capacitation medium or in media added with 2.4 µM olygomycin A. : Control cells. : Spermatozoa incubated in capacitation medium added with 2.4 µM olygomycin A from the begin‐ ning of the incubation. ▲: Spermatozoa incubated in a standard capacitation medium for 4h and subsequent added with 10 µg/mL progesterone and 2.4 µM olygomycin A together. B): Sperm cells incubated in a standard capacitation medium or in Ca2+-depleted media. ○: Spermatozoa incubated in capacitation medium without Ca2+ and added with 2 mM EGTA from the beginning of the experiments. : Spermatozoa incubated in a standard capacitation medium for 4h and subsequent added with 10 µg/mL progesterone and 2 mM EGTA together Results are expressed as means ±S.E.M. for 7 separate experiments. Asterisks indicate significant (P<0.05) differences when compared with the respec‐ tive Control values. Results excerpted from [43]) and unpublished data from our laboratory.

However, as exposed above, monosaccharides are not the only energy source that sperm can utilize. Other substrates, such as citrate and lactate, can be utilized to obtain energy, at least in several mammalian species. The ways by which mammalian sperm utilize these nonmonosaccharide substrates have not been as thoroughly studied as those linked to monosac‐ charide metabolization. In this way, boar sperm have been one of the most studied species. In this species, extracellular citrate and lactate are utilized after their intake by metaboliza‐ tion through the Krebs cycle [37]. This metabolization is the same than that detailed for many other cellular types. However, sperm utilization of citrate and lactate has several specific features. Thus, a sperm-specific lactate dehydrogenase (LDH) isozyme has been described in several species [12, 27, 37, 40, 41]. This specific isozyme, named LDH-X is the most important LDH form in sperm in which it has been described, such as boar [27], whereas its activity presents several differentiate features. In this sense, the LDH-X is distributed in both soluble and non-soluble fractions of sperm extracts obtained through sonication [37], indicating thus the existence of a specific distribution pattern of this LDH-X in sperm. More‐ over, the kinetic characteristics of the LDH are different, depending on the location of the enzyme, either in the soluble or the non-soluble sperm extract fraction [37]. In fact, immu‐ nocytochemistry of boar sperm has shown that the LDH-X is mainly located at the mid‐ piece and principal area of the tail, linking thus its activity to the neighboring of mitochondrialocated Krebs cycle activity [37]. All of these information clearly indicate that the regulation of sperm LDH activity, and hence lactate metabolism, is regulated in a very complex man‐ ner, with mechanisms depending on factors such as the precise location of the key regula‐ tory enzymes. Another interesting feature of both sperm lactate and citrate metabolism is that lactate enters the Krebs cycle through a direct pathway, which does not need its previ‐ ous conversion to pyruvate [27, 37]. This direct pathway is important, since it not only produces energy, but also relevant levels of reductive potential, allowing sperm to regener‐ ate significant amounts of NAD+ . Regarding citrate, sperm can metabolize it through two simultaneous pathways. The first pathway is through direct utilization by Krebs cycle, yield‐ ing CO2 and ATP. The second pathway is indirect, by following two sequential steps. A first step in which citrate enters into the Krebs cycle. In the second step the metabolites de‐ rived from citrate after its pass through the Krebs cycle are directed to the pyruvate carbox‐ ylase step, which converted these metabolites in lactate, which, in turn, will be sent to the extracellular medium and again re-entered into the Krebs cycle through the LDH-X step. At first glance, the biological meaning of this second, convoluted pathway is not immediately understood. However, if the maintenance of a correct NAD+ /NADH equilibrium is consid‐ ered as basic to maintain a proper sperm function, the main objective of this second, indi‐ rect pathway would be not the obtainment of energy, but of reductive potential. In this way, citrate and lactate can have a paramount role not as energy producers, but as reductive potential metabolites.

glucose [33, 39)]. This pre-eminence of glycolysis in species like boar and mouse arises to an important question, if sperm mitochondria seem no have a predominant role in these spe‐ cies in obtaining energy, what are their main role? Investigators can only speculate on this point, although there are several data regqarding mainly boar sperm that can aid to obtain a better vision of this issue. The first data correspond to the observation of boar sperm mitochon‐ dria ultra-structure (Figure 3). Electron microscope images of boar sperm mitochondria show an organella with very few prominent inner membrane crests. Instead of this, the inner mitochondrial space is mainly occupied by thin and short crests and with an amorphous and homogeneous matrix. This is very different to the classical image for mitochondria, which, like those form hepatocytes, show an inner structure crowded with prominent inner crests. Taking into account that the most important steps of the electronic transport system and subsequent ATP synthesis are structurally linked to inner mitochondrial crests, it is easy to assume that boar sperm mitochondria would be not be very efficient as energy suppliers. In fact, the oxygen consumption rate of boar sperm, which is a direct measure of mitochondri‐ al ability to generate energy, is about 2 magnitude orders lower than that measured in pig hepatocytes [4, 43]. However, this does not preclude that mitochondria-originated energy would not be important for sperm function in species in which glycolysis is the most impor‐ tant energy-synthesizing pathway. Regarding this point, our laboratory has shown that the achievement of a feasible, progesterone-induced "in vitro" acrosome reaction is concomitant with a sudden and intense peak of O2 consumption rate and also of intracellular ATP levels ([43] and Figures 2,4). Furthermore, unpublished data from our laboratory clearly shows that this peak is not present in conditions in which progesterone-induced acrosome reaction is prevented. These results strongly suggest the existence of a close relationship between mito‐ chondria-generated energy and the achievement of the acrosome reaction, despite of the low

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**Figure 3.** Ultrastructural image of boar sperm mitochondria. The low development of inner crests is noticeable (aster‐ isks). BM: inner mitochondrial membrane. P: cell membrane. A: axoneme. FD: dense fibres. GP: peripheral granules.

energy-efficiency of these organelles.

From [7]).
