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BOR - Papers in Press, published online ahead of print June 23, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029041
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BIOLOGY OF REPRODUCTION 71, 1437–1445 (2004)
DOI: 10.1095/biolreprod.104.029041
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Gluconeogenesis-Linked Glycogen Metabolism Is Important in the Achievement of In Vitro Capacitation of Dog Spermatozoa in a Medium Without Glucose1

J.L. Albarracín3, J.M. Fernández-Novell4, J. Ballester3, M.C. Rauch5, A. Quintero-Moreno3, A. Peña3, T. Mogas3, T. Rigau3, A. Yañez5, J.J. Guinovart4, J.C. Slebe5, I.I. Concha5, and J.E. Rodríguez-Gil2,3

Unit of Animal Reproduction,3 Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain Department of Biochemistry and Molecular Biology and IRBB,4 Scientific Park of Barcelona, University of Barcelona, Barcelona, Spain Instituto de Bioquímica,5 Facultad de Ciencias, Campus Isla Teja, Universidad Austral de Chile, Valdivia, Chile


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [14C]glycogen after the addition to l-CCM with [14C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.

dog sperm, gamete biology, gluconeogenesis, glycogen metabolism, in vitro capacitation, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Capacitation of mammalian spermatozoa is a functional process that requires the consumption of significant amounts of energy. Thus, the changes in motion parameters, cell-membrane composition, and the increase and variations of tyrosine phosphorylation (Tyr-Phos) patterns are all energy consuming [1, 2]. However, the energy source needed to maintain capacitation, at least in some mammalian species, seems to be highly specific. Thus, glucose seems to be the major energy source needed in maintaining in vitro capacitation in mice and human spermatozoa because this sugar induces much higher penetration rates and capacitation-like changes than other monosaccharides, like fructose or mannose [3, 4]. In fact, the study of changes in the chlorotetracycline (CTC) patterns after in vitro capacitation of mice spermatozoa has indicated that these changes are glucose dependent [3]. All of these results have indicated, too, that mammalian spermatozoa must have a sophisticated metabolic system that allows them to specifically differentiate the energy susbtrates taken by the cells in order to render specific changes in their functionality.

Previous studies in our laboratory have shown that dog sperm has the ability to use separate sugars in order to obtain distinct functional results. Thus, dog spermatozoa have shown specific changes in motility and Tyr-Phos patterns, depending on the presence of either fructose or glucose in the medium [5, 6]. These changes seem to be related to the presence of specific systems that allow one to differentiate among sugars, such as the presence of the specific hexose transporters GLUT 3 and GLUT 5, which are placed in separate locations of the cell, or the existence of both fructose-specific and glucose-specific hexokinase activities, also located in separate zones of dog sperm [6]. Moreover, dog-sperm energy metabolism is very complex because there are many metabolic pathways where hexoses can be diverted. In this sense, these cells not only have the glycolysis and Krebs cycle catabolic pathways but also the glycogen synthesis and pentose phosphate cycle [6, 7]. Furthermore, the regulation of these anabolic pathways seems to be very complex and, in the case of glycogen synthesis, this is controlled not only by changes in the key enzymatic activities of the pathway, but also in displacements of these enzymes, especially glycogen synthase, throughout the cell [8]. All of this portrays a very complex system that allows for a fine regulation of energy levels in dog spermatozoa depending on their functional status.

In vitro capacitation of dog spermatozoa can be achieved with ease by incubating the cells in different capacitation media. In this sense, several papers have shown this point, monitoring in vitro capacitation by several functional tests, like changes in CTC patterns, evaluation of acrosome reaction, and variations in motility patterns [9, 10]. In the majority of cases, the capacitation medium for dog sperm contained glucose, and a glucose-free medium seems to not be effective in inducing capacitation [9, 10]. However, this result was contradictory with that observed in our laboratory, in that in vitro capacitation of dog spermatozoa can also be feasibly achieved after incubation of cells in a capacitation medium without sugars and containing only lactate and pyruvate as the energy source [11]. At first glance, this is contradictory in the case where the presence of a glycolytic substrate, such as glucose as the essential energy substrate to attain capacitation, was considered. Thus, substituting glucose for three-carbon compounds as energy substrates could not exert the same specific effects leading to capacitation. One possibility to overcome this contradiction was that dog sperm be able to obtain glucose from these three-carbon compounds and, in this way, this newly synthesized sugar could induce its capacitating effects. Nevertheless, this would imply the existence of a gluconeogenic pathway in dog spermatozoa. This would be in sharp contrast with practically all that is known on sperm energy metabolism, which has been classified as primordially glycolytic [12]. However, because dog sperm present some fully functional anabolic pathways, as described above [6, 7], it would be of the greatest importance to test for the existence of a putative gluconeogenic pathway in these cells.

The main aim of this work is to test the possibility that dog spermatozoa have a fully functional gluconeogenic pathway, in which they can obtain glucose from substrates such as L-lactate. To this end, several metabolite markers of dog sperm, like intracellular glycogen levels, CO2 formation, and the presence of the gluconeogenic enzymes, fructose 1,6-bisphosphatase (FBPase) and aldolase B, were analyzed in cells subjected to capacitation in a medium without glucose, as in [11]. Moreover, dog semen was subjected to capacitation in the l-CCM medium in the absence or the presence of a specific inhibitor of the entry of L-lactate to gluconeogenesis, phenylacetic acid (PAA), in order to establish the effects of this inhibition in the achievement of in vitro capacitation in the l-CCM medium. Our results indicate that dog sperm is able to synthesize glucose from lactate besides utilizing this three-carbon compound to obtain energy through the Krebs cycle. The newly formed glucose is mainly stored as glycogen. Finally, gluconeogenesis has a noticeable role in the achievement of in vitro capacitation in the absence of glucose because its inhibition altered capacitation markers of dog sperm, such as Tyr-Phos patterns, motion parameters, or CTC stain location.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All procedures described within were reviewed and approved by the Autonomous University of Barcelona Animal Care and Use Committee and were performed in accordance with the Animal Welfare Law issued by the Catalan Government (Generalitat de Catalunya, Spain).

Animals and Sample Collection

Semen was obtained from 10 purebred Beagle dogs aged 3–6 yr. The dogs were kept in outdoor kennels, exercised twice daily, and fed a balanced diet with free access to water. Semen was collected once or twice weekly, without using a female, by manual stimulation into warmed (37°C) sterile glass or plastic funnels. Only the sperm-rich fraction of each ejaculate was used.

In Vitro Capacitation Procedure

In vitro capacitation was carried out by taking the procedure described in [11] as a basis. For this, 3–4 sperm-rich fractions were pooled. At this time, an aliquot was taken to perform the required analyses. Immediately thereafter, pooled samples were washed three times by dilution in a Krebs-Ringer-Henseleit medium (pH 7.4) without any monosaccharide, as in [6], and a further centrifugation at 200 x g for 10 min. After the last washing, the sperm was resuspended in the capacitation medium (l-CCM medium) at a final concentration of 60–80 x 106 sperm/ml. Incubation in the l-CCM medium was maintained for 4 h at 38.5°C in a 5% CO2 atmosphere. At the end of the incubations, aliquots were taken to perform the appropriate analyses. When stated, aliquots of samples were also taken after only 1 h or 2 h of incubation. The l-CCM medium was composed of 83.5 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 37.6 mM NaHCO3, 0.25 mM sodium pyruvate, 21.55 mM sodium lactate, and the final addition of 0.4% (v/w) bovine serum albumin (Sigma, St. Louis, MO). The osmolarity of the l-CCM medium was 301 ± 5 mOsm (mean ± SEM), and its pH was 7.8.

Processing of Samples and Determination of Glycogen Content, [14C] Lactate Incorporation to Glycogen, and Glycogen Immunolocalization

To determine intracellular glycogen levels, dog-sperm aliquots incubated in l-CCM at the indicated times were collected, immediately centrifuged at 2000 x g for 30 sec, and the resultant pellets were then frozen in liquid N2 and stored at –80°C until their use. Glycogen was quantified enzymically, as described in [7].

The [14C] incorporation to glycogen from [14C] lactate was analyzed following the technique described by Chan and Exton [13], which demonstrated the specificity of alcoholic precipitation for glycogen determination. Following this, dog samples were incubated in a l-CCM medium that contained 21.55 mM [U-14C] lactate (total specific radioactivity: 0.3 µCi/sample). One-milliliter aliquots of sperm suspension in [14C]-marked l-CCM were taken at the indicated times, centrifuged at 2000 x g for 30 sec, and the resultant pellets were resuspended in 250 µl of 30% (w/v) KOH. These resuspended samples were heated at 100°C for 15 min, and the resultant sperm homogenate was placed onto small squares (about 0.25 mm2) of 31-ET Whatman filter paper (Whatman, Maidstone, UK). At this moment, a 10-µl aliquot was taken in order to determine the total protein content of the samples. When the filter paper absorbed all of the sample, glycogen was precipitated onto it by immersion in an ice-cold 66% ethanol solution. Precipitated papers were washed three times in successive 66% ethanol solutions to eliminate non-glycogen-associated radioactivity, and finally they were dried in a microwave oven. The [14C] counts included in the paper were analyzed in a liquid scintillation counter.

Glycogen immunolocalization was performed as described in [7] after using a monoclonal, specific antiglycogen antibody that has been shown to bind specifically to glycogen from chondrocytes, hepatocytes, muscle cells, and dog spermatozoa, as well as to purified glycogen [7]. Previous to performing glycogen immunolocalization, samples were stained with Hoechst 33258 fluorescent staining (Sigma) in order to better determine precise location of glycogen in sperm. The complete sequence to carry out this Hoechst 33258/glycogen double staining was the following.

At the indicated times, sperm samples were seeded onto gelatin-coated glass slides (26 x 76 mm). Then, the slides were covered with a phosphate-buffered saline solution (PBS; pH 7.4) containing 0.1% (v/v) commercial Hoechst 33258 solution. Incubation was carried out at 38.5°C for 15 min, preventing any light source from reaching the slides. After this, the excess liquid on the coverslips was eliminated by decantation, and the slides were thoroughly washed three times with PBS. The stained spermatozoa were further fixed for 30 min in PBS containing 4% paraformaldehyde, and after this, they were again thoroughly washed another three times with PBS. Glycogen immunocytochemistry was started by incubating with 1 mg/ml NaBH4 in order to eliminate autofluorescence. This step was followed by a permeabilization with 0.2% (w/v) Triton X-100 in PBS and a blocking step in 3% bovine serum albumin/PBS (w/v). Spermatozoa were then incubated with the antiglycogen antibody (dilution 1/500, v/v) for 1 h at 15°C, washed with PBS, and treated with a tetramethylrhodamine isothiocyanate-conjugated swine anti-rabbit immunoglobulin (DAKO, Glostrup, Denmark). Fluorescent images were obtained with a Leica TCS 4D confocal scanning microscope (Leica Lasertechnik, Heidelberg, Germany) adapted to an inverted Leitz DMIRBE microscope and a 63x (NA 1.4 oil) Leitz Plan-Apo Lens (Leitz, Stuttgart, Germany). The light source was an argon/krypton laser (75 mW). Final, arbitrary colors shown here (red and green) were chosen in order to obtain a better contrast between both stains. Successive confocal slices of the images (image thickness: from 0.5 to 1 µm) were integrated in order to obtain three-dimensional spermatozoa images, which were further stored as TIFF-format images. These images were simultaneously observed and stored under visible light and a phase-contrast microscope. The combination of visible light and laser images allows for the exact location of spermatozoa that present a positive reaction to both the Hoechst stain and the glycogen immunolocalization, as well as for the exact location of the signals within spermatozoa.

Immunological Detection of Gluconeogenic Enzymes and Tyrosine Phosphorylation Pattern

For immunoperoxidase localization, spermatozoa were spread onto slides, fixed in buffered paraformaldehyde-acetone, treated with 0.3% H2O2 for 15 min and incubated for 60 min at room temperature in 5% BSA-PBS, pH 7.4, followed by incubation overnight at 4°C with anti-FBPase and antialdolase purified according to [14, 15]. Spermatozoa were washed and incubated with anti-rabbit IgG-horseradish peroxidase (1:100, Amersham Corporation, Arlington Heights, IL) for 2.5 h at room temperature. Immunostaining was developed using 0.05% 3,3'-diaminobenzidine and 0.03% H2O2. As controls, spermatozoa were incubated with the antibody used and preadsorbed with the respective proteins used to generate it.

On the other hand, the immunoblotting location of gluconeogenic enzymes and the Tyr-Phos pattern was performed as follows.

Spermatozoa membrane proteins, which were used to determine the presence of gluconeogenic enzymes, were obtained as previously described [16], whereas samples utilized to detect the Tyr-Phos pattern were homogenized by sonication and further treated as described in [6]. In both cases, samples were then resolved by SDS-PAGE (30 µg per lane) in 10% polyacrylamide gel [17], followed by transfer to a nitrocellulose membrane (Protran; Schleicher and Schuell, Dasell, Germany) as in [18]. The transferred samples were tested with the antibodies at a dilution (v/v) of 1/ 2000 (aldolase B and FBPase) and 1/1000 (PY-20 anti-Tyr-Phos antibody; Chemicon International, Temecula, CA). Immunoreactive proteins were tested using peroxidase-conjugated anti-rabbit secondary antibody (Amersham, Buckinghamshire, UK), and the reaction was developed by chemoluminescence (Amersham).

Recording In Vitro Capacitation of Dog Spermatozoa in Canine Capacitation Medium

The appearance of in vitro capacitation in dog sperm after incubation in l-CCM was determined after the combined, global evaluation of the following parameters (see [11]).

Determination of the percentages of viability and altered acrosomes For this, aliquots were taken after 0 h (control), 1 h, 2 h, 3 h, and 4 h of incubation in l-CCM and were then subjected to the dual Trypan Blue/ Giemsa vital staining [19]. In this technique, viable cells show an overall grayish-light blue color, whereas nonviable sperm have either a partial or total dark-blue staining. On the other hand, the presence of an intact, regular acrosomal ridge under the Trypan Blue/Giemsa staining was indicative of the presence of intact acrosomes, whereas any alteration of this acrosomal ridge was interpreted as a structurally altered acrosome. Following this, our results indicated that the incubation in the l-CCM induces a progressive decrease of viability concomitantly to an increase in the percentage of altered acrosomes. However, the decrease of viability was significant (P < 0.05) only at 4 h of incubation, whereas the increase in altered acrosomes was significant (P < 0.05) only after 3 h of incubation (Fig. 1). Thus, viability ranged from 90.0% ± 1.0% in control samples to 68.0% ± 6.0% after 4 h of incubation, whereas the percentage of altered acrosomes went from 4.5% ± 0.5% in the control to 11.0% ± 1.0% after 4 h of incubation (Fig. 1). This indicates a progressive decline in the overall vitality of the sperm, which was significant after 3–4 h of incubation.



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  		FIG. 1. Effects of incubation in l-CCM in the absence or presence of 10 mM phenylacetic acid on the percentages of viability and altered acrosomes of dog sperm. Incubation of dog sperm in the l-CCM was conducted at 38.5°C in a 5% CO2 atmosphere, as described in the Materials and Methods section. Figures show the percentages of viability (A) and altered acrosomes (B) of dog sperm incubated at the indicated times in the l-CCM medium with (black circles) or without 10 mM phenylacetic acid (black triangles). Results are expressed as means ± SEM of eight separate experiments. Asterisks indicate a significant (P < 0.05) difference, with respect to the 0-h values, whereas crosses indicate significant (P < 0.05) differences between samples incubated with or without phenylacetic acid

Determination of motion patterns Aliquots were taken at the times indicated above and were then subjected to a computer-assisted system analysis of motility (CASA), like that described in [5]. For this purpose, a 5-µl aliquot was placed on a prewarmed (37°C) siliconized microslide and covered with a 25 mm2 siliconized coverslip. Observations were made at a magnification of 200x on a negative phase-contrast microscope with a warmed stage (37°C). In the CASA system, four fields per sample were analyzed, and the total number of spermatozoa analyzed in each semen sample (including those not motile) ranged from 20 to 50. In the case of sperm incubated for 4 h in the l-CCM medium, motility analysis was performed only in those cells that were not aggregated themselves, and in this case, 20–50 individual sperm were also analyzed. Our CASA system was based on the analysis of 16 consecutive, digitalized photographic images obtained from a single field. These 16 consecutive photographs were taken in a time-lapse of 0.64 sec, which implied a velocity of image-capturing of one photograph each 40 msec. The sperm-motility descriptors obtained after CASA and utilized in this study are the following.

Curvilinear velocity (VCL) The instantaneously recorded sequential progression along the entire trajectory of the spermatozoon. Its units are µm/sec.

Linear velocity (VSL) The straight trajectory of the spermatozoon per unit of time. Its units are µm/sec.

Mean velocity (VAP) The mean trajectory of the spermatozoon per unit of time. Its units are µm/sec.

Linear coefficient (LIN) The mathematical relationship (VSL/VCL) x 100. Its units are %.

Straightness coefficient (STR) The mathematical relationship (VSL/ VAP) x 100. Its units are %.

Wobble coefficient (WOB) The mathematical relationship (VAP/VCL) x 100. Its units are %.

Mean amplitude of head lateral displacement (mean ALH) The mean head displacement along its curvilinear trajectory around the mean trajectory. Its units are µm.

Dance (DNC) The mathematical relationship VCL x mALH. Its units are µm2/sec.

Mean harmonic oscillation of the head (HME) The mean value of the distance between the curvilinear trajectory, with respect to the mean trajectory. Its units are µm.

Frequency of head displacement (BCF) The number of lateral oscillatory movements of the sperm head around the mean trajectory. Its units are Hz.

Total motility was defined as the percentage of spermatozoa with a linear velocity (VAP) above 20 µm/sec, and the above-described motion parameters were only determined in spermatozoa with a VAP ≥ 20 µm/ sec. Results indicated that dog sperm from control samples showed high percentages of total motility (87.2% ± 4.3%; Table 1). This value declined slightly after 2 h of incubation, but at 3–4 h, a great change in motion parameters was observed. In this respect, after 4 h of incubation, total motility showed a sharp decline (6.3% ± 0.3%; see Table 1), which was induced by the appearance of head-to-head sperm agglutination. Head-agglutinated sperm showed a highly characteristic flagellar movement, with low frequency and high amplitude. Nonagglutinated spermatozoa showed a motility pattern with values of VCL, VSL, VAP, mean ALH, DNC, and HME significantly (P < 0.05) higher than those observed in fresh samples (Table 1). These results were consistent with the appearance of hyperactivated spermatozoa, whereas the head-to-head, agglutinated spermatozoa would be sperm with achieved capacitation [11]. Thus, these results are compatible with the appearance of a functional in vitro capacitation after 3–4 h of incubation in l-CCM.


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  		TABLE 1. Effects of incubation in 1-CCM for 4 h in the absence or pres ence of 10 mM phenylacetic acid on the motility parameters of dog sper matozoa.*

Determination of the Tyr-Phos pattern Dog-sperm extracts from fresh ejaculates showed the presence of a faint, specific pattern of Tyr-Phos with phosphorylated proteins in the range of about 30 kDa to roughly 100 kDa (Fig. 2). Incubation for 2 h in l-CCM showed the same phosphorylation pattern, although a noticeable increase in the intensity of phosphorylation was detected (Fig. 2). Finally, there was a dramatic change in the Tyr-Phos pattern after 4 h of incubation in l-CCM. In this case, the overall intensity of reaction was much more intense than in fresh samples. Moreover, there was a great increase in the phosphorylation of several proteins in a range of about 30 kDa to approximately 50 kDa, the majority of which were not detected in fresh ejaculates (Fig. 2). This increase in the phosphorylation and change in the specific Tyr-Phos pattern has been defined as a marker for dog-sperm capacitation [11, 20], and it has been taken in this sense in our experiments.



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  		FIG. 2. Effects of incubation in l-CCM in the absence or presence of 10 mM phenylacetic acid on the tyrosine phosphorylation pattern of dog sperm. Western blot procedure and incubation of dog sperm in the l-CCM were carried out as indicated in the Materials and Methods section. 0 h, Fresh dog-sperm samples; 2 h, cells incubated in l-CCM for 2 h; 4 h, sperm incubated in l-CCM for 4 h. –PAA, Cells incubated without phenylacetic acid (PAA); + PAA, sperm incubated in the presence of 10 mM PAA; M, molecular weight markers. C–, samples treated without the primary antibody as negative controls of the blot. Figure shows a representative Western blot of five separate experiments

Determination of the chlorotetracycline pattern As indicated above, a change in the CTC marking location has been described as specific after in vitro capacitation of dog sperm [10, 21]. Following this, we observed the appearance of this change in our in vitro capacitation conditions. For this purpose, the CTC stain was carried out as in [10, 21]. Briefly, sperm samples were centrifuged at 200 x g for 10 min, and the resultant cellular pellet was mixed with 45 µl of a daily prepared 20-mM Tris and 5-mM cysteine buffer (T/C buffer; pH 7.8) containing 750 µM CTC. The cells were incubated in this solution for 20 sec, and after this, they were spread onto a clean microscope slide. These slides were covered with a 2% (w/ v) paraformaldehyde solution in freshly prepared T/C buffer and then 25 µl of 1,4-diaza-2,2,2-bicyclo-octane (DABCO; Sigma) was also spread onto the slides to attenuate the fading of fluorescence. After adding a coverslip, the slides were compressed to eliminate any excess liquid. The coverslip was finally sealed with colorless nail polish, and the slides were stored at 4°C in the dark until their microscope observation. As before, fluorescent images were obtained with a Leica TCS 4D confocal scanning microscope (Leica Lasertechnik), adapted to an inverted Leitz DMIRBE microscope and a 63x (NA 1.4 oil) Leitz Plan-Apo Lens (Leitz). The light source was an argon/krypton laser (75 mW). Successive confocal slices of the images (image thickness: from 0.5 to 1 µm) were integrated in order to obtain three-dimensional spermatozoa images, which were further stored as TIFF-format images. These images were simultaneously observed and stored under visible light and a phase-contrast microscope, in order to precisely locate those spermatozoa that reacted to the CTC stain.

The above-described technique showed that fresh dog sperm had an intense CTC location at midpiece (Fig. 3). Moreover, CTC staining was also observed in the head, although its intensity of stain was less (Fig. 3). This pattern, observed in practically all of the stained cells, changed after 4 h of incubation in the l-CCM medium, when there was a general, uniform, and slight CTC staining in both midpiece and head in practically all of the marked cells. The uniformity of the results meant that no quantitative statistical analysis of these changes was performed with this test (Fig. 3).



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  		FIG. 3. Effects of incubation in l-CCM in the absence or presence of 10 mM phenylacetic acid on the chlorotetracycline location pattern of dog sperm. The figure shows representative pictures from five separate experiments of dog spermatozoa from fresh samples (A) and incubated in l-CCM for 4 h in the absence (B) or presence of 10 mM phenylacetic acid (C). D) Negative control taken with the same values of brightness and contrast as those of the positive images. Arrows indicate midpiece and asterisks indicate sperm head. Original magnification is x650

Following all four parameters, only those samples that showed all of the above-described changes in a simultaneous manner after incubation in the l-CCM medium were considered in this work.

Statistical Techniques

The evaluation of putative statistically significant differences among the experimental groups was tested, in parametrical parameters, such as those of motion, by using the PROC GLM procedure included in the SAS statistical package [21]. For this purpose, the experimental groups and the day of performing the experiments were considered as fixed parameters. When the PROC GLM procedure detected any statistically significant (P < 0.05) differences, a LSMEANS procedure, also included in the SAS package, was further applied to perform the corresponding means comparisons. To identify putative statistical differences in nonparametrical analyses, namely percentages of viability and altered acrosomes, a chi-square test, also included in the SAS package, was applied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Glycogen Metabolism after In Vitro Capacitation of Dog Sperm in the l-CCM Medium

In vitro capacitation of dog semen in the l-CCM medium induced changes in the sperm glycogen content. Thus, fresh samples showed intracellular glycogen levels of 0.48 ± 0.07 nmol glucose/106 sperm (mean ± SEM for eight separate experiments; Fig. 4). These values progressively increased with incubation and reached a significant (P < 0.05) peak after 2 h of incubation in l-CCM (0.81 ± 0.09 nmol glucose/106 sperm; Fig. 4). After this, glycogen progressively decreased, reaching values of 0.37 ± 0.07 nmol glucose/106 sperm after 4 h of incubation (Fig. 4). Compatible results were observed when the incorporation of [U-14C] lactate to glycogen was determined, although in this case, [14C] incorporation to glycogen was practically constant during the incubation with l-CCM after up to 4 h (Table 2).



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  		FIG. 4. Effects of incubation in l-CCM on the intracellular glycogen levels of dog sperm. Incubation of dog sperm in l-CCM was conducted at 38.5°C in a 5% CO2 atmosphere, as described in the Materials and Methods section. The figure shows the intracellular glycogen levels of dog sperm incubated at the indicated times in the l-CCM medium (black triangles). Results are expressed as means ± SEM of eight separate experiments. Asterisks indicate a significant (P < 0.05) difference, with respect to the 0-h values


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  		TABLE 2. Effects of incubation in 1-CCM in the absence or presence of 10 mM phenylacetic acid on the [U-14C] lactate incorporation to glycogen of dog sperm.*

The decrease of glycogen content after a peak at 2 h of incubation with l-CCM could be a consequence of a physiological glycogenolytic process initiated after the first 2 h of incubation that was glycogenic, or it could also be related to the significant decrease in vitality after 3–4 h of incubation with l-CCM. In the latter case, the decrease of glycogen could be due to the death of a significant percentage of cells, whereas live sperm that suffers capacitation conserved elevated amounts of glycogen. In this way, the loss of glycogen induced by a physiological glycogenolysis would render the image of spermatozoa with a uniform loss of glycogen markings, whereas a decrease of glycogen concomitant with a process of cellular death would show a heterogeneous image of live spermatozoa with a maximal amount of glycogen because glycogen synthesis in live cells would continue for up to 4 h, together with dead sperm cells that lacked any signal of the polysaccharide. In order to elucidate this point, an immunolocalization of sperm glycogen in cells subjected simultaneously to a staining with Hoechst 33258 was carried out. Results indicated that dog sperm from fresh ejaculates accumulated the polysaccharide mainly in the postacrosomal zone of the head and the midpiece, as published in [8] (Fig. 5). After 2 h of incubation with l-CCM, glycogen was mainly located in the midpiece, and the glycogen signal maintained its intensity in a uniform manner in all cells that contained the polysaccharide (data not shown). Sperm subjected to incubation with l-CCM for 4 h showed a more or less uniform appearance in those cells that had appreciable amounts of immunolocated glycogen (Fig. 5). In this case, the glycogen accumulation in the midpiece was faint to very faint, although the immunological signal of the polysaccharide was uniform in those cells that presented a positive signal against the specific antiglycogen antibody (Fig. 5). These results suggest that the decrease of glycogen content after 2 h of incubation in l-CCM was mainly associated with a physiological glycogenolytic process associated with the incubation in this medium rather than with a sperm death-related process of glycogen loss.



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  		FIG. 5. Effects of incubation in l-CCM on glycogen location of dog sperm. The figure shows representative pictures from five separate experiments of dog spermatozoa from fresh samples (A) and incubated in l-CCM for 4 h at 38.5°C in a 5% CO2 atmosphere (B). C) Negative control taken with the same values of brightness and contrast as those of the positive images. Arrows indicate midpiece and asterisks indicate sperm head. Original magnification is x400

Finally, no trace of extracellular glucose was detected after incubation in l-CCM for up to 4 h, neither after enzymatic analysis of the medium nor by finding extracellular [14C]-glucose (data not shown).

Presence of Gluconeogenic Enzymes in Dog Spermatozoa

One of the key regulatory enzymes of the gluconeogenic and glyconeogenic pathways is FBPase (EC 3.1.3.11), which catalyzes the irreversible conversion of fructose 1,6-bisphosphate (Fru 1,6-P2) to fructose 6-phosphate (Fru 6-P) and inorganic phosphate [22–24]. In addition, fructose 1,6-bisphosphatase aldolase (aldolase; EC 4.1.2.13) catalyzes the reversible conversion of Fru 1,6-P2 to glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate and participates both in gluconeogenesis and glycolysis [25, 26]. On the basis of the kinetic properties and subcellular localization of the aldolase isoenzymes, it has been suggested that aldolase B, expressed predominantly in the liver, has evolved to have a role in gluconeogenesis, while aldolase A, the classic muscle isoenzyme, is more effective participating in glycolysis [14, 27–29]. Following this, the presence of FBPase and aldolase B in dog spermatozoa was therefore tested. Immunoblotting of membrane proteins extracted from dog spermatozoa revealed that, for both antibodies, reactivity was limited to one immunoreactive protein band (Fig. 6). The FBPase antibody reacted with a strongly immunoreactive protein band that migrated with an apparent molecular mass of approximately 40 kDa (Fig. 6A). In a similar manner, aldolase B antibody reacted with a single protein band with an apparent molecular mass of approximately 39 kDa (Fig. 6B). The size of the immunoreactive bands corresponded to the size of the respective bands obtained from liver protein extracts (Fig. 6C). Additionally, no changes were observed after incubation with l-CCM (Fig. 6). No immunoreactive protein bands were observed when the antibodies were preabsorbed with the proteins used to elicit them, thus confirming the specificity of the reaction (data not shown).



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  		FIG. 6. Detection of the fructose 1,6-bisphosphatase and aldolase B proteins in dog spermatozoa. Proteins isolated from dog spermatozoa were fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, transferred to immobilon membranes, and probed with anti-FBPase (A) and antialdolase B (B) antibodies, followed by incubation with a secondary antibody coupled to peroxidase. 0 h, Fresh dog-sperm samples; 2 h, cells incubated in l-CCM for 2 h; 4 h, sperm incubated in l-CCM for 4 h. Rat-liver proteins were used as positive controls (C); anti-FBPase (lane 1) and antialdolase B (lane 2) antibodies. Sizes on the right are kDa and indicate the migration of molecular mass standards. The figure shows a representative Western blot of three separate experiments

Dog spermatozoa from fresh ejaculates showed positive immunoreactivity with FBPase antibody, with intense immunolabeling observed in the acrosomal zone of the head and the tail (Fig. 7A). Aldolase B was mainly present in the equatorial zone and midpiece of the tail (Fig. 7B). The specificity of the reaction was confirmed in experiments that showed an absence of immunoreaction when preabsorbed antibodies were used (Fig. 7, A' and B'). No great changes in the localization of these enzymes were observed after incubation with l-CCM (data not shown).



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  		FIG. 7. Immunolocalization of fructose 1,6-bisphosphatase and aldolase B in dog spermatozoa. Dog spermatozoa were spread onto coated slides and probed with anti-FBPase (A) and antialdolase B (B) antibodies, followed by incubation with a secondary antibody conjugated with horseradish peroxidase. No reactivity was observed with preabsorbed antibodies (A' and B', respectively). Original magnification, x900

Effects of Phenylacetic Acid on Glycogen Metabolism of Dog Sperm Incubated in the l-CCM Medium

Phenylacetic acid (PAA) is a specific inhibitor of gluconeogenesis from lactate by inhibition of the pyruvate carboxylase reaction [30]. The addition of 10 mM PAA to the l-CCM medium almost completely prevented [14C] lactate incorporation to glycogen. Thus, this incorporation went, after 2 h of incubation, from 0.11 ± 0.01 nmol glucose/106 sperm in control cells to 0.03 ± 0.01 nmol glucose/106 sperm in the l-CCM medium with 10 mM PAA added, whereas similar results were observed in the rest of the tested times (Table 2).

Effects of Phenylacetic Acid on In Vitro Capacitation Markers of Dog Sperm Incubated in the l-CCM Medium

Next, the effects that the addition of PAA would have on three markers of in vitro capacitation, as described in the Material and Methods, were tested: the percentages of viability and altered acrosomes, the motion characteristics, the Tyr-Phos pattern, and the CTC location pattern.

The addition of 10 mM PAA to l-CCM did not greatly affect the percentage of viability after up to 4 h of incubation. Only significant (P < 0.05) differences were determined between viability values of fresh samples and those incubated for 4 h both with and without the addition of PAA (Fig. 1). However, PAA did seem to affect the percentage of altered acrosomes in some way, although the significant (P < 0.05) effects were concentrated in a clear increase of altered acrosomes after incubation in the presence of 10 mM PAA during the first 2 h (Fig. 1). On the other hand, 10 mM PAA caused a complete disappearance of progressive spermatozoa, which was indicated by values of 0% of total motility, after 3–4 h of incubation (Table 1; data not shown). Thus, in these samples, the only observed movement was that of agglutinated spermatozoa, which showed flagellar movements similar to those of samples without PAA (Table 1; data not shown).

The addition of PAA also had a profound effect on the Tyr-Phos pattern of dog-sperm extracts subjected to l-CCM incubation. Thus, as shown in Figure 2, incubation for 2 h in the presence of 10 mM PAA prevented the increase of phosphorylation observed in control samples. This effect was even more pronounced, and in some cases, reached a total lack of Tyr-Phos after 4 h of incubation. Moreover, PAA prevented the change in the Tyr-Phos pattern observed after 4 h of incubation in control cells, in those cases where phosphorylation was detected (Fig. 2).

Similarly, PAA affected the CTC location pattern, although it was not possible to exactly quantify the precise percentage of spermatozoa that showed each separate type of CTC marking locations. Notwithstanding, the practically universal, uniform, and slight staining observed after 4 h of incubation in l-CCM was altered by the appearance of heterogeneous CTC location patterns, with cells showing faint, granulated CTC distribution in the midpiece and postacrosomal zone of the head, some others with CTC accumulated in the midpiece and other cells, which showed no CTC marking (Fig. 3). It is also noteworthy that the CTC pattern observed after 4 h of incubation with l-CCM was different from that determined in fresh samples (Fig. 3).

In conclusion, the global interpretation of the effects of PAA on all of the tested markers (percentages of viability and altered acrosomes, motility characteristics, Tyr-Phos pattern, and CTC location) indicates that the inhibition of gluconeogenesis alters, rather than prevents, the induction of an in vitro capacitation in the l-CCM medium.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data clearly indicate that dog sperm has a fully functional gluconeogenic pathway from substrates like L-lactate. Moreover, these results also show that this newly synthesized glucose has some relevance in the maintenance of in vitro capacitation when this process is induced in a medium without an external source of sugar. This is evident after a global analysis of the alterations in in vitro capacitation markers associated with the inhibition of gluconeogenesis in cells incubated in the l-CCM medium. Thus, dog-sperm gluconeogenesis is not only a functional process, but it can also play an important role in maintaining sperm functionality under specific conditions.

The statement that gluconeogenesis is functional in dog sperm is based on the following points. First, there was a clear glycogen synthesis in cells incubated in the l-CCM medium and, second, by the presence of at least two of the key enzymes that regulate gluconeogenesis, FBPase and aldolase B [14, 22–24, 27–30]. Focusing on glycogen synthesis, it might be possible that the increase in glycogen content observed in the first 2 h of incubation could be due to the use of other endogenous substrates to form this glycogen. In this sense, some mammalian sperm, such as boar, can use endogenous substrates, like glycerol, as energy sources. However, in our case, the experiments with [U-14C] lactate and the comparison between the rate of [14C]-marked substrate incorporation and the clear intracellular glycogen-level increase in the first 2 h clearly demonstrate that practically all of the newly formed glycogen came from an exogenous source. It is also striking that, although [14C]-marked lactate can be found in an intracellular store, it has not been found in the form of extracellular glucose. Thus, the final product of dog-sperm gluconeogenesis is not extracellular glucose. This is easily explained by the lack of glucose 6-phosphatase activity of dog sperm, as has previously been described [7]. A consequence of this fact is that dog sperm utilizes gluconeogenesis as a source to obtain self-consuming energy, which can be immediately directed to the precise needs of spermatozoa at any given time.

Our results support the hypothesis of a close relationship between glycogen metabolism and the attainment of in vitro capacitation. Thus, an increase of glycogen levels during the first 2 h of incubation was observed, followed by a progressive decrease after that. It is noteworthy that the initiation of the signs that mark in vitro capacitation of dog sperm has been established at around 2–3 h of incubation in a capacitation medium [9, 10, 18]. This would indicate that the starting of glycogenolysis would coincide with the establishment of capacitation. A feasible interpretation of this result is that dog sperm accumulates glycogen, which will be needed as the energy source for all of the capacitation-dependent mechanisms that will be activated after 2 h of incubation. Because the majority of these mechanisms are energy consuming [1, 2], the existence of a rapid and feasible energy source will be of importance in their correct attainment. Furthermore, the observed alteration of capacitation markers like Tyr-Phos and CTC patterns when dog sperm is incubated in the presence of PAA indicates that the glycogenesis/further glycogenolysis sequence is relevant for the proper achievement of in vitro capacitation. In this way, the gluconeogenesis-linked, sequential glycogen metabolism is important in the establishment of capacitation under our conditions. On the other hand, the important role of glycogen would be at the basis of the contradictory results published earlier about the achievement of a functional in vitro capacitation in the absence of glucose [9– 11] because the presence of low glycogen levels in fresh samples or the lack of a rapid glycogenic rhythm could impede the attainment of capacitation. Thus, any alteration of glycogen metabolism, which would be more intense in the absence of sugars because glycogenesis is more pronounced in the presence of glucose or fructose [7, 8] than with lactate/pyruvate, could induce the described lack of positive results.

The presence of a functional gluconeogenic pathway that is important in the attainment of in vitro capacitation in the absence of sugars is a striking phenomenon not described until now. In fact, other species, such as boar [32] or bull [33] show a total lack of this metabolic pathway. This indicates the existence of a metabolic specialization among mammalian sperm. We suggest that this specialization could be related to distinct survival strategies that each mammalian species has developed for its spermatozoa. Thus, dog sperm shows a remarkably high survival rate inside the bitch genital tract, in contrast with other species, such as boar [34, 35]. This could be related to the fact that the energy metabolism of dog sperm shows itself to be very complex, with a highly sophisticated network of regulatory mechanisms. This complexity, which includes the existence of metabolic pathways, like the pentose phosphate cycle and a very active glycogen metabolism [6, 7], contrasts with other, much simpler, in metabolic terms, species like boar and bull [32, 33], which, as stated above, concomitantly have a much shorter life-span inside the female genital tract [35, 36]. Remarkably, both boar and bull sperm necessarily need the presence of sugars such as glucose in the medium to achieve in vitro capacitation, and we suggest that the lack of gluconeogenesis in these species leads to this necessity. In fact, all of the results indicate that glucose is a necessary requirement to attain in vitro capacitation in all mammalian sperm, although the origin of the sugar can be from exogenous sources or, in dog, from an endogenous and autogenous origin. In all events, we hypothesize that the specific functionality of sperm from a single mammalian species is strongly related to its specific metabolic characteristics because parameters like the specific motility characteristics and the life-span inside the female genital tract are strongly linked to energy-management mechanisms. Thus, we state that metabolic specialization is an important feature to understand functional specialization of mammalian sperm.

In summary, our results clearly indicate that dog sperm is able to synthesize glucose and, hence, accumulate it in the form of glycogen from gluconeogenic precursors like lactate. Moreover, gluconeogenesis-linked glycogen metabolism is important to regulate the achievement of in vitro capacitation in the absence of glucose, suggesting that, although glucose is instrumental in the attainment of dog sperm in vitro capacitation, the origin of this sugar can be from endogenous sources. Finally, the very sophisticated regulatory network of energy management could be at the basis of the very specific functional characteristics observed in dog spermatozoa.


   ACKNOWLEDGMENTS
 
We wish to thank Mr. Chuck Simmons for his valuable assistance in preparing the English version of this manuscript.


   FOOTNOTES
 
1 Partially supported by research grants from the Fondo Nacional de Ciencia y Tecnologí (Chile), FONDECYT 1010720, and from the Dirección de Investigación y Desarrollo (Universidad Austral de Chile), DID-UACH 200205. Back

2 Correspondence: Joan E. Rodríguez-Gil, Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain. FAX: 34 935812006; juanenrique.rodriguez{at}uab.es Back

Received: 27 February 2004.

First decision: 23 March 2004.

Accepted: 14 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Roldán ERS. Signal transduction during mammalian sperm acrosomal exocytosis. In: Lauria A, Gandolfi F, Enne G and Gianaroli L (eds.), Gametes: Development and Function. Rome: Serono Symposia; 1988: 219–228
  2. Visconti PE, Kopf GS. Regulation of protein phosphorylation during sperm capacitation. Biol Reprod 1998 59:1-6[Free Full Text]
  3. Fraser LR, Herod JE. Expression of capacitation-dependent changes in chlorotetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycosylable substrate. J Reprod Fertil 1990 88:611-621
  4. Rogers BJ, Perreault SD. Importance of glycosylable substrates for in vitro capacitation of human spermatozoa. Biol Reprod 1990 43:1064-1069[Abstract]
  5. Rigau T, Farré M, Ballester J, Mogas T, Peña A, Rodríguez-Gil JE. Effects of glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculates. Theriogenology 2001 56:801-815[CrossRef][Medline]
  6. Rigau T, Rivera M, Palomo MJ, Fernández-Novell JM, Mogas T, Ballester J, Peña A, Otaegui PJ, Guinovart JJ, Rodríguez-Gil JE. Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa. Reproduction 2002 123:579-591[Abstract]
  7. Ballester J, Fernández-Novell JM, Rutllant J, García-Rocha M, Palomo MJ, Mogas T, Peña A, Rigau T, Guinovart JJ, Rodríguez-Gil JE. Evidence for a functional glycogen metabolism in mature mammalian spermatozoa. Mol Reprod Dev 2000 56:207-219[CrossRef][Medline]
  8. Palomo MJ, Fernández-Novell JM, Peña A, Guinovart JJ, Rigau T, Rodríguez-Gil JE. Glucose- and fructose-induced dog-sperm glycogen synthesis shows specific changes in the location of the sperm glycogen deposition. Mol Reprod Dev 2003 64:349-359[CrossRef][Medline]
  9. Mahi CA, Yanagimachi R. Capacitation, acrosome reaction, and egg penetration by canine spermatozoa in a simple defined medium. Gamete Res 1978 1:101-109
  10. Rota A, Peña AI, Linde-Forsberg C, Rodríguez-Martínez H. In vitro capacitation of fresh, chilled and frozen-thawed dog spermatozoa assessed by the chlorotetracycline assay and changes in motility patterns. Ann Reprod Sci 1999 57:199-215
  11. Albarracín JL, Mogas T, Palomo MJ, Peña A, Rigau T, Rodríguez-Gil JE. In vitro capacitation and acrosome reaction of dog spermatozoa can be feasibly attained in a defined medium without glucose. Reprod Domest Anim 2004 39:1-7[CrossRef][Medline]
  12. Mann T. Biochemistry of semen. In: Greep RO, Astwood EB (eds.), Handbook of Physiology. Washington, DC: American Physiology Society; 1975:321–347
  13. Chan TM, Exton JH. A rapid method for the determination of glycogen content and radioactivity in small quantities of tissue or isolated hepatocytes. Anal Biochem 1976 71:96-105[CrossRef][Medline]
  14. Sáez D, Slebe JC. Subcellular localization of aldolase B. J Cell Biochem 2000 78:62-72[CrossRef][Medline]
  15. Yañez A, Nualart F, Droppelmann C, Brito M, Concha II, Slebe JC. Localization of the key gluconeogenic enzymes fructose 1,6-bisphosphatase, and the phosphoenolpyruvate carboxy kinase in different tissues. J Cell Physiol 2003 197:189-197[CrossRef][Medline]
  16. Angulo C, Rauch MC, Droppelmann A, Reyes AM, Slebe JC, Delgado-López F, Guaiquil VH, Vera JC, Concha II. Hexose transporter expression and function in mammalian spermatozoa: cellular localization and transport of hexoses and vitamin C. J Cell Biochem 1998 71:189-203[CrossRef][Medline]
  17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
  18. Burnett WN. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated proteins. J Anal Biochem 1981 112:195-203[CrossRef][Medline]
  19. Rodríguez-Gil JE, Montserrat A, Rigau T. Effects of hypoosmotic incubation on acrosome and tail structure on canine spermatozoa. Theriogenology 1994 42:815-829
  20. Petrunkina AM, Simon K, Gunzel-Apel AR, Topfer-Petersen E. Specific order in the appearance of protein tyrosine phosphorylation patterns is functionally coordinated with dog sperm hyperactivation and capacitation. J Androl 2003 24:423-437[Abstract/Free Full Text]
  21. Guérin P, Ferrer M, Fontbonne A, Bénigni L, Jacquet M, Ménézo Y. In vitro capacitation of dog spermatozoa as assessed by chlortetracycline staining. Theriogenology 1999 52:617-628[CrossRef][Medline]
  22. SAS. SAS/STAC Software. Cary, NC; SAS Institute, Inc.; 1996
  23. Hers HG, Francois J, Van Schaftingen E. Fructose 2,6-bisphosphate versus cyclic AMP in the liver and in lower eukaryotic cells. Curr Top Cell Regul 1985 27:399-410[Medline]
  24. Pilkis SJ, Granner DK. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol 1992 54:885-909[CrossRef][Medline]
  25. Tejwani GA. Regulation of fructose-bisphosphatase activity. Adv Enzymol 1983 54:121-124
  26. Morse DE, Horecker BL. The mechanism of action of aldolases. Adv Enzymol 1968 31:125-181
  27. Penhoet EE, Rutter NJ. Detection and isolation of mammalian fructose diphosphate aldolases. Methods Enzymol 1975 42:240-249[Medline]
  28. Lebherz HG, Rutter WJ. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry 1969 8:109-121[CrossRef][Medline]
  29. Penhoet EE, Kochmann M, Rutter NJ. Molecular and catalytic properties of aldolase C. Biochemistry 1969 8:4396-4402[CrossRef][Medline]
  30. Eagles PA, Iqbal M. A comparative study of aldolase from human muscle and liver. Biochem J 1973 133:429-439[Medline]
  31. Bahl JJ, Matsuda M, DeFronzo RA, Bressler R. In vitro and in vivo suppression of gluconeogenesis by inhibition of pyruvate carboxylase. Biochem Pharmacol 1997 53:64-67
  32. Marin S, Chiang K, Bassilian S, Lee W-NP, Boros LG, Fernández-Novell JM, Centelles JJ, Medrano A, Rodríguez-Gil JE, Cascante M. Metabolic strategy of boar spermatozoa revealed by a metabolic characterization. FEBS Lett 2003 554:342-346[CrossRef][Medline]
  33. Hammersted RH, Lardy HA. The effects of substrate cycling on the ATP yield of sperm glycolysis. J Biol Chem 1983 258:8759-8768[Abstract/Free Full Text]
  34. Feldman EC, Nelson RW. Fertilization. In: Pedersen E (ed.), Canine and Feline Endocrinology and Reproduction. Philadelphia: WB Saunders Co.; 1987: 420–421
  35. Hunter RHF. Interrelationships between spermatozoa, the female reproductive tract and the egg investments. In: Cole DJA, Foxcroft GR (eds.), Control of Pig Reproduction. London: Butterworth Scientific; 1982:49–64
  36. Salisbury GW, VanDemark NL, Lodge JR. Sperm survival into the female reproductive tract. In: Salisbury GW, VanDemark NL, Lodge JR (eds.), Physiology of Reproduction and Artificial Insemination of Cattle. San Francisco: W.H. Freeman and Co.; 1978:394–396



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