**5. Roles of mitochondria in the control of the overall mature boar sperm function**

As previously indicated, the main energy source for mature mammalian sperm are ATPs obtained either through the glycolytic pathway or mitochondrial oxidative pathways. The precise equilibrium between both energy-obtaining pathways will be different among spe‐ cies and in cases like boar and mice, this equilibrium is greatly unbalanced towards glycoly‐ sis, which is the overly majoritary energy-obtaining pathway in the presence of sugars like 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 Furthermore, unpublished data from our laboratory clearly shows that this peak is not present in conditions in which progesterone-induced acrosome reaction is prevented.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 energy-efficiency of these organelles.

monosaccharide 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‐

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

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

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

**5. Roles of mitochondria in the control of the overall mature boar sperm**

As previously indicated, the main energy source for mature mammalian sperm are ATPs obtained either through the glycolytic pathway or mitochondrial oxidative pathways. The precise equilibrium between both energy-obtaining pathways will be different among spe‐ cies and in cases like boar and mice, this equilibrium is greatly unbalanced towards glycoly‐ sis, which is the overly majoritary energy-obtaining pathway in the presence of sugars like

. Regarding citrate, sperm can metabolize it through two

/NADH equilibrium is consid‐

ate significant amounts of NAD+

potential metabolites.

**function**

understood. However, if the maintenance of a correct NAD+

**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. From [7]).

**Figure 4.** Intracellular ATP levels of boar sperm subjected to "in vitro" capacitation and subsequent "in vitro" acro‐ some 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 beginning 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 respective Control values. Results excerpted from unpublished data from our laboratory.

**Figure 5.** Percentages of total motility of boar sperm subjected to "in vitro" capacitation and subsequent "in vitro" acrosome 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. Total motility has been defined as the percentage of spermatozoa with a curvilinear velocity (VCL) higher than 20 µm/sec. 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 be‐ ginning of the incubation. ▲: Spermatozoa incubated in a standard capacitation medium for 4h and subsequent add‐ ed 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

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However, the fact that Krebs cycle seems to be important only in punctual moments of the boar sperm life-time does not necessarily indicate that boar sperm mitochondria are only important in this point. It is noteworthy that mitochondria have much more roles than purely

the respective Control values. Results excerpted from 42) and unpublished data from our laboratory

**Figure 5.** Percentages of total motility of boar sperm subjected to "in vitro" capacitation and subsequent "in vitro" acrosome 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. Total motility has been defined as the percentage of spermatozoa with a curvilinear velocity (VCL) higher than 20 µm/sec. 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 be‐ ginning of the incubation. ▲: Spermatozoa incubated in a standard capacitation medium for 4h and subsequent add‐ ed 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 respective Control values. Results excerpted from 42) and unpublished data from our laboratory

**Figure 4.** Intracellular ATP levels of boar sperm subjected to "in vitro" capacitation and subsequent "in vitro" acro‐ some 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 beginning 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 respective

Control values. Results excerpted from unpublished data from our laboratory.

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

However, the fact that Krebs cycle seems to be important only in punctual moments of the boar sperm life-time does not necessarily indicate that boar sperm mitochondria are only important in this point. It is noteworthy that mitochondria have much more roles than purely being a mere energy-producing factory. Mitochondria also play a key role in the control of other very important aspects of eukaryotic cells function, like modulation of apoptosis and the control of calcium metabolism. Thus, it is very probable that mitochondria from sperm of species like boar or mouse exert their most important functions on other cellular function‐ al points than energy management. Unpublished results from our laboratory are strongly pointing out this supposition. Thus, the incubation of boar sperm in a capacitation medi‐ um in the presence of olygomycin A, a specific inhibitor of the electronic chain and the chemiosmosis steps [11], immobilizes boar sperm and prevent them to achieve "in vitro" capacitation. However, this effect was accomplished without any significant changes in the rhythm of O2 production and the intracellular ATP levels (Figures 2, 4, 5 and data not shown from our labvoratory). In contrast, the incubation of boar sperm in a capacitation medium without calcium induces an increase in the velocity parameters of these cells, although the achievement of capacitation is also prevented (data not shown). The effect linked to the lack of extracellular calcium however, is again concomitant with no changes in both the rhythm of O2 production and the intracellular ATP levels (Figures 2, 4 and data not shown). The conclusion from these results is that boar (and probably mice) sperm mitochondria play an important regulatory role in the control of functional aspects such as motility patterns and the achievement of "in vitro" capacitation by ways that are not directly linked to energy production. This opens a new perspective in the manner in which investigators would have to approximate to the understanding of the mitochondria role in the control of sperm func‐ tion. However, much more work is needed in order to achieve a complete view of this complex phenomenon.

serve would play an important role in the maintenance of "in vivo" sperm survival. In fact, as discussed above, the existence of a fully functional glycogen metabolism has been dem‐ onstrated in sperm from species like dog, boar, horse and ram [5]. Remarkably, dog sperm shows the most active glycogen metabolism of all of the studied species, in this way accu‐ mulating the maximal recorded intracellular levels [5]. As described above, this glycogen plays an important role in the achievement of feasible "in vitro" capacitation [1, 2], reinforc‐ ing thus the importance of this anabolic pathway in dog. The importance of glycogen syn‐ thesis in dog would be surely linked to another important feature, also described above. It is worth noting that dog sperm is the only studied species so far that shows the presence of two separate hexokinase activities. The first of them is similar to hexokinase-I, which is present in all of the studied mammalian sperm. The second, however, is similar in kinetic and immunologic properties to the hepatic and pancreatic isoform glucokinase [16]. The presence of a glucokinase-like activity in dog sperm but not in other species like boar ac‐ quires utmost importance when the precise role that hepatic and pancreatic glucokinase plays is studied. Thus, it is well known that hepatic glucokinase acts as a "metabolistate" that diverts hexoses metabolism to either anabolic or catabolic pathways, depending on fac‐ tors such as the precise physiologic cell status and sugar extracellular levels [9]. If a similar role for the dog sperm glucokinase-like activity is assumed, the inference that this protein also regulates the entry of energy metabolites in either anabolic or catabolic pathways can be also yielded. These assumptions, notwithstanding will depend on both the precise energy necessities and the extracellular concentrations of sugars inside the female genital tract. Moreover, this "metabolistate" seems to be in the basis of above described, observed differ‐ ential effects of fructose and glucose in the serine phosphorylation levels of dog sperm pro‐ teins like protein kinase C [17]. Thus, dog sperm reaches an even more fine regulation of not only their intracellular energy levels, but also their overall functional status. This very fine

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regulation would surely increase survival ability of these cells.

mal conditions.

These two separate metabolic phenotypes would not surely be the only present among mammalian species. Much more work is needed in order to describe and analyze this phe‐ nomenon. In any case, the existence of these metabolic phenotypes would be of the greatest importance. These phenotypes, in fact, will be the reflection of the sperm specialization due to the adoption of separate reproductive strategies among mammals. Thus, these separate evolutive, reproductive strategies will cause the existence of great differences among sperm of separate species not only under a morphological, but also under a metabolic point of view. These differences among species would be, in turn, at the basis of the described differ‐ ences in vital aspects of sperm function, such as motility patterns and capacitation mecha‐ nisms. Finally, these physiological differences would also be reflected in changes in the specific strategy developed to store a particular semen sample from a precise species in opti‐
