energy metabolism and its linkage to intracellular ca2+ and ph regulation in rat spermatogenic cells

Original article

Energy metabolism and its linkage to intracellular Ca2+ and pHregulation in rat spermatogenic cells

Estefania Herreraa, Karime Salasb, Nestor Lagosb, Dale J. Benosc, Juan G. Reyesa,c*a Instituto de Quimica, Universidad Catolica de Valparaiso, Casilla 4059, Valparaiso, Chile

b Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile

c Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, AL35294-0005, USA

Received 16 May 2000; accepted 14 July 2000

Energy metabolism and intracellular adenine nucleotides of meiotic and postmeiotic spermatogenic cells arehighly dependent on external substrates for oxidative phosphorylation and glycolysis. Using fluorescentprobes to measure the changes in cytosolic [Ca2+] ([Ca2+]i) and pH (pHi), we were able to demonstrate thatchanges in energy metabolism of meiotic and postmeiotic spermatogenic cells were rapidly translated intochanges of pHi and [Ca2+]i in the absence or presence of external Ca2+. Under these conditions, mitochondriawere gaining cytosolic calcium in these cells. Our results indicate that Ca2+ mobilised by changes inmetabolic energy pathways originated in thapsigargin-sensitive intracellular Ca2+ stores. Changes inintracellular adenine nucleotides, measured by HPLC, and a likely colocalization of ATP-producing andATP-consuming processes in the cells seemed to provide the linkage between metabolic fluxes and thechanges in pHi and [Ca2+]i in pachytene spermatocytes and round spermatids. Glucose metabolismproduced an increase of [Ca2+]i in round spermatids but not in pachytene spermatocytes, and a decrease inpHi in both cell types. Hence, glucose emerges as a molecule that can differentially modulate [Ca2+]i and pHiin pachytene spermatocytes and round spermatids in rats. © 2000 Éditions scientifiques et médicalesElsevier SAS

bafilomycin / seminiferous tubules / spermatogenesis / testis

1. INTRODUCTION

Growth and differentiation of spermatogenic cellsoccur under the influence and control of Sertoli cells inthe seminiferous tubule (Jegou, 1993). Meiotic andpostmeiotic spermatogenic cells rely mainly on oxida-tive ATP production for their energy metabolism

(Reyes et al., 1990). The substrate for oxidative metabo-lism in these cells has been proposed to be lactate,produced from glucose by Sertoli cells (Robinson andFritz, 1981; Mita and Hall, 1982). FSH and isoproter-enol stimulated glucose consumption and lactate pro-duction in Sertoli cells (Mita et al., 1982; Le Gac et al.,1983; Hall and Mita, 1984). The location of hexosetransporters in the seminiferous tubule (Angulo et al.,1998) suggests that glucose, together with lactate, canbe secreted into the adluminal compartment of theseminiferous tubule. These metabolites would then be

* Correspondence and reprints: fax: +56 32 273 422.E-mail address: [email protected] (J.G. Reyes).

Biology of the Cell 92 (2000) 429−440© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0248490000010820/FLA

[Ca2+]i, pHi and spermatogenic cell energy metabolism Herrera et al.

taken up and metabolised by spermatogenic cells (Jutteet al., 1981, Mita et al., 1982, Nakamura et al., 1986).Glucose metabolism induced a net ATP hydrolysis inround spermatids by activation of a substrate cycle inphosphofructokinase/fructose bisphosphatase. Hence,the hormone-dependent supply of energy substratesby Sertoli cells would regulate the metabolic energyfluxes and nucleotide pools in meiotic and postmeioticspermatogenic cells. This metabolic connection hasbeen proposed to be part of the signals by whichSertoli cells regulate spermatogenic cell developmentin the seminiferous tubules. However, the mechanismsby which changes in energy metabolism could modu-late spermatogenic cell functions and developmenthave not been explored.

Intracellular Ca2+ concentration ([Ca2+]i) and intrac-ellular pH (pHi) can modulate many cell functions,including cell cycle and differentiation in eukaryoticcells (Madshus, 1988, Whitaker and Patel, 1990). In ratround spermatids, the steady-state [Ca2+]i was main-tained by both plasma membrane and intracellularCa2+-ATPases (Berrios et al., 1998). Similarly, intracel-lular pH (pHi) was maintained mainly by the action ofa plasma membrane V-type H+–ATPase (Osses et al.,1997). Although the roles of pHi and [Ca2+]i in sper-matogenic cell development have not been studied, theimportance of the H+ and Ca2+ ATP-dependent trans-port systems in [Ca2+]i and pHi homeostasis in sper-matogenic cells suggested to us that these intracellularparameters could be linking the energy state and meta-bolic energy fluxes of the cells with other cell functionsaffected by [Ca2+]i and pHi.

In this work, we explored the hypothesis that [Ca2+]iand pHi are cell parameters that respond to the ener-getic state and the metabolic energy fluxes in ratpachytene spermatocytes and round spermatids. Be-cause the energy metabolism of these two types ofspermatogenic cells differ, suggesting that it could bedifferentiation-dependent, we also explored the hy-pothesis that pachytene spermatocyte and round sper-matid [Ca2+]i and pHi respond differently to thechanges in metabolic energy fluxes induced by lactateor glucose. For these purposes, we used fluorescentprobes to measure [Ca2+]i and pHi in spermatogeniccells. In order to correlate the changes of adeninenucleotides with the changes in intracellular Ca2+ andpH homeostasis, we measured the cellular content ofATP, ADP and AMP in spermatocytes and round sper-matids using HPLC. The possible anatomical associa-tion of ATP producing organelles (mitochondria) andATP-dependent transport systems were analysed byvital location of mitochondria, plasma membrane andendoplasmic reticulum (ER). Our results show that[Ca2+]i and pHi in these cells were sensitive to changesin mitochondrial and glycolytic metabolism. [Ca2+]iand pHi in round spermatids are more sensitive than in

pachytene spermatocytes to the provision of externalglucose. The mechanisms linking mitochondrial me-tabolism and transport ATPases are not sensitive tookadaic acid treatment. Topological correlations be-tween mitochondria and ATP-dependent transport sys-tems are likely, to facilitate active H+ transport in ratspermatid plasma membrane and ER Ca2+ transport inpachytene spermatocytes. However, no evident ana-tomical correlation was found for plasma membraneH+ transport in pachytene spermatocytes, or ER Ca2+

transport in round spermatids. Besides anatomical as-sociation of some of the transport systems with mito-chondria in each cell type, adenine nucleotideregulation of transport ATPases seems the most likelyconnection linking energy metabolism and [Ca2+]i andpHi regulation in round spermatids and pachytenespermatocytes. Mitochondria can act as part of thenetwork of [Ca2+]i homeostatic mechanisms modulatedby energy metabolism in spermatogenic cells.

2. MATERIALS AND METHODS

2.1. Rat spermatogenic cell preparation

Rat spermatogenic cell populations were preparedfrom the testicles of adult (60 days old) Wistar rats asdescribed by Romrell et al. (1976). The pachytenespermatocyte (85 ± 5% purity, 17 ± 2 µm, range15–20 µm) and round spermatid fractions (92 ± 4% pu-rity, 11 ± 2 µm, range 8–13 µm) were identified both bytheir size, as well as by the typical aspect of theirnucleus stained with Hoechst-33342 (Reyes et al.,1997). The contaminating cells in the pachytene sper-matocyte fraction were smaller size cells (spermatids50%, residual bodies 20%, and leptotene and zygotenespermatocytes 30% of the contaminating cells). Becauseof the large size of pachytene spermatocytes, theirvolume was calculated to be approximately 96% of thetotal cell volume in this fraction. The contaminatingcells in the round spermatid fraction were spermato-cytes and residual bodies (20 and 80% of the contami-nating cells, respectively). The calculated volume ofround spermatids corresponds to approximately 94%of the total cell volume in this fraction. The integrity ofthe cell membrane was estimated incubating the cellsuspension with 4 µM ethidium bromide and examina-tion under fluorescence microscopy. In all the condi-tions tested in this work, the cell membrane integritywas > 90%.

2.2. Intracellular Ca2+ measurementsof spermatogenic cells in suspension

Measurements of [Ca2+]i in pachytene spermatocytesand round spermatids in suspension were performedin cells loaded with fura-2. Cells were loaded with the

430 Biology of the Cell 92 (2000) 429–440


dye by incubation of approximately 1 mg·mL–1 cellproteins (5 × 106 cells·mL–1) with 5 µMacetoxymethyl–fura-2 for 1 h at room temperature andin a 95% air/5% CO2 atmosphere. The cells were thenwashed three times in KH medium. The measurementswere performed in a PTI Deltascan fluorimeter using aratiometric method as described by Grynkiewicz et al.(1985). Calibration of fura-2 was performed by lysis ofthe cells with digitonin (20 µg·mL–1) in the mediumthat contained 0.5 mM Ca2+(Fmax) and subsequent ad-dition of a final concentration of 5 mM EGTA, pH 7.4(Fmin).

The dose–response of thapsigargin (tsg, a specificinhibitor of sarco-endoplasmic reticulum Ca2+[SERCA]ATPases) on [Ca2+]i homeostasis was estimated fromthe initial rate of [Ca2+]i rise after tsg addition to thecells in suspension. These measurements were per-formed in the absence of external Ca2+ (3 nM) in afluorimeter cuvette with temperature control (33 °C).

2.3. Intracellular pH measurements

The intracellular pH of rat pachytene spermatocytesand round spermatids was estimated from fluores-cence measurements of intracellular 2’,7’–bis–(2–carboxyethyl)–5(and–6)carboxy fluorescein (BCECF)(Rink et al., 1982). Cells were loaded with BCECF byincubation in KH–lactate with 1 µM acetoxymethyl–B-CECF (30 min at 34 °C in a 95% air/5% CO2 atmo-sphere). The cells were washed three times in KHmedium and resuspended at a concentration of ap-proximately 1 mg·mL–1 cell proteins. Observation ofthe cells under epifluorescence microscopy showed ahomogeneous distribution of the dye throughout thecytoplasm. Intracellular BCECF was calibrated in ahigh potassium medium plus nigericin (10 mg·mL–1)and the excitation fluorescence ratio 505/445 nm wascalculated at different extracellular pH values (Rink etal., 1982).

The dose–response curve for the effect of bafilomy-cin A (bafA, an inhibitor of V-type H+–ATPases) on pHihomeostasis was estimated from the initial rate of pHidecrease after bafA addition to the cell suspension inthe fluorimeter cuvette at 33 ºC.

2.4. Determination of the levelsof intracellular adenine-nucleotides in ratround spermatids

Rat round spermatids in suspension were preincu-bated at a concentration of approximately 6 mg·mL–1

cell proteins in KH Ca2+-lactate in a 95% air/5% CO2atmosphere for 10 min. Subsequently, different experi-mental additions were made. After the reported incu-bation times, 400 µL of the cells were added to an icecold microcentrifuge tube and pelleted by centrifuga-tion for 30 s at 16 000 × g. The supernatant was then

removed, 200 µL of ice cold 0.5 mM EGTA, pH 7.2 wasadded and the cells were vortexed for 10 s. Addition of50 µL of 3.2 M perchloric acid and maintenance in icecold bath for 30 s was followed by a 1 min centrifuga-tion. 200 µL of the perchloric acid supernatant wasmixed with 200 µL of KOH/Hepes/0.15M KCl ad-justed to give a final pH of 7.2–7.4. A procedure andstorage control was made by taking duplicate 200 µLsamples of a 2 mM ATP solution and subjecting themto the previously mentioned sample processing andstorage conditions. The samples were maintained fro-zen at –20o C until defrosted and analysed by an HPLCequipped with high pressure pumps coupled to auto-mated gradient controller, UCK injector, and UV detec-tor (model 501, Millipore-Waters, Bedford, MA, USA).Nucleotide separation was obtained with an anionexchange column (MA7Q, 50 × 7.8 mm, Bio-Rad,Hercules, CA, USA) at a flow of 1.5 mL·min–1 and atroom temperature. The mobile phases were0.01 M MOPS + 0.01 M KCl, pH 7.0 and 0.1 M MOPS+ 0.5 M KCl, pH 7.0. Calibration was performed withdiluted 2 mM standards of ATP, ADP and AMP.

2.5. Vital location of spermatogenic cellmitochondria and endoplasmic reticulum

Rat spermatogenic cells were separately loaded with1 µM MitoTracker Red or ER-Tracker Blue–White DPXfor 30 min at room temperature in KH–lactate buffer.The cells were deposited in a microscope chamber andwashed by perifusion. The cells labelled with Mi-toTracker Red were exposed to 4 µM Hoescht 33342,observed and selected using the characteristics of theirnucleus (Reyes et al., 1997). Serial confocal images(1 µm optical depth) were obtained on a Zeiss LSM 410confocal inverted microscope using a 100 × oil immer-sion objective. The cells labelled with ER Tracker wereobserved in a Nikon Diaphot microscope with 100 × oilimmersion objective and digital images were takenwith a Spectrasource cooled CCD camera. The ERimages were digitally corrected for out of focus lightusing a no-neighbors correction algorithm (Monck etal., 1992). After the ER images were taken, the cellswere permeabilized with 20 µg·mL–1 digitonin, ex-posed to 4 µM ethidium bromide and the characteris-tics of their nucleus were recorded to identify the typeof spermatogenic cell observed.

2.6. Statistical analysis

The data were analysed using ANOVA followed by aBonferroni test. Where indicated, a paired t test wasperformed in order to document relative differences ina variable. Non-linear regression was performed withthe ORIGIN software package. The values reported aremean ± SD unless stated otherwise.

Biology of the Cell 92 (2000) 429–440 431


2.7. Chemicals

MitoTracker Red CM-H2Xros, ER-Tracker Blue–White DPX, BODIPY TR ceramide,2’,7’–bis–(2–carboxy ethyl)–5(and 6)carboxy fluorescein(BCECF) and fura-2 acetoxymethyl esters were ob-tained from Molecular Probes (Eugene, OR, USA).N,N’-dicyclohexylcarbodiimide (DCCD), carbonyl cya-nide m–chlorophenyl-hydrazone (CCCP), antimycin,rotenone, oligomycin and all the other enzymes, saltsand buffers were obtained from Sigma Chem. Co. (StLouis, MO, USA). Thapsigargin (tsg) and bafilomycinA (bafA) were obtained from Calbiochem (La Jolla, CA,USA).

3. RESULTS

3.1. Effects of mitochondrial energymetabolism on [Ca2+]i homeostasis in ratspermatogenic cells

Rat round spermatids [Ca2+]i in lactate-containingmedia in the presence or absence of extracellular Ca2+

(0.5 mM) and at 33 ºC were 30 ± 10 nM (SD, n = 22) and19 ± 9 nM (n = 57), respectively (significantly different,p < 0.001). In similar conditions, pachytene spermato-cytes presented values of [Ca2+]i of 42 ± 14 nM (SD, n =4) and 18 ± 3 nM (n = 3) in the presence or absence ofexternal Ca2+, respectively (significantly different,p < 0.01).

When rat spermatids were deprived of externalenergy substrates for 20 min at 33 ºC in a 95% air/5%CO2 atmosphere in the absence of external Ca2+ (1 mMEGTA, approximately 3 nM free Ca2+), they progres-sively gained cytosolic Ca2+ (figure 1A). Similar resultswere observed in the presence of external calcium(0.5 mM, not shown). Addition of lactate produced areversal of the increase in [Ca2+]i within 15 s. After-ward, addition of oligomycin, an inhibitor of the mito-chondrial H+–ATP synthase, produced a rapid increasein [Ca2+]i. A comparable effect was observed withantimycin (5 µM, an inhibitor of the ubiquinone–cyto-chrome c oxidoreductase electron chain complex),KCN (1 mM, an inhibitor of the cytochrome c–O2oxidoreductase electron chain complex) and DCCD(50 µM, a nonspecific inhibitor of H+–ATPases) (notshown). Thus, round spermatids [Ca2+]i respond rap-idly to changes in mitochondrial energy metabolism.

Similar effects were observed with pachytene sper-matocytes (figure 1A), except that these cells presenteda less marked gain in [Ca2+]i in the absence of externalsubstrates and a faster recovery of the basal levels of[Ca2+]i after lactate addition as compared to rat sper-matids (figure 1A).

Thus, intracellular Ca2+ stores in round spermatidsand pachytene spermatocytes respond rapidly to

changes in mitochondrial metabolism by taking up orreleasing Ca2+ to the cytosol. The gain in cytosolic Ca2+

came from tsg-sensitive intracellular Ca2+ stores (ICaS),since a saturating dose of tsg added after a steady state

Figure 1. A. Intracellular levels of Ca2+ in rat pachytene sperma-tocytes (open circles) and round spermatids (closed circles)loaded with fura-2. The cells were previously incubated at 33 ºC inthe absence of extracellular energy substrates for 20 min in a95% air/5% CO2 atmosphere. At the indicated times 1 mML-lactate (pH 7.4) or 5 µM oligomycin was added to the cellsuspension. The data shown are representative of measurementsperformed in four different cell preparations with similar results.B. Dose–response curves for the thapsigargin-induced initial rateof Ca2+ release from intracellular stores in rat pachytene sperma-tocytes (open circles) and round spermatids (closed circles)loaded with fura-2. The initial rate of Ca2+ release from intracellu-lar stores was estimated by linear regression of the [Ca2+]ichanges between 5 and 45 s after thapsigargin addition to thecells.



oligomycin increase in [Ca2+]i produced only a minorchange in [Ca2+]i (approximately 10 ± 5 nM, n = 3). TheCa2+ released by oligomycin did not come from amitochondrial compartment, since in these conditionsround spermatid mitochondria appear to take up Ca2+

(see below).In the absence of external Ca2+, the increase in

cytosolic Ca2+ could originate either from a decreasedrate of Ca2+ uptake by ICaS, or from an increased rateof release from these compartments. However, addi-tion of tsg (300 nM, causing a complete inhibition ofSERCA ATPases) followed by oligomycin (5 s later)induced an initial rate of rise in [Ca2+]i that was notsignificantly different from tsg alone (n = 3, notshown). The absence of an additive effect of oligomy-cin and tsg strongly suggest that the main effect ofoligomycin was an inhibition of the rate of uptake bySERCA ATPases in spermatogenic cells.

In order to estimate what fraction of the maximalrate of Ca2+ release from ICaS (obtained with a saturat-ing dose of tsg) was obtained with oligomycin, wedetermined the dose–response curve for the tsg-induced initial velocity of Ca2+ release from ICaS (inthe absence of external Ca2+). As shown in figure 1B, inround spermatids tsg presented a hyperbolic dose–re-sponse curve with a K0.5 = 44 nM. Oligomycin (5 µM)produced an effect (0.48 ± 0.12 nM·min–1, n = 4),equivalent to approximately 25% of the maximal rateof tsg-induced Ca2+ release. In pachytene spermato-cytes, the dose–response curve showed a sigmoidshape with a K0.5 = 45 nM. Oligomycin produced aneffect (0.31 ± 0.14 nM·min–1, n = 4) equivalent to ap-proximately 16% of the maximal rate of tsg-inducedCa2+ release. Thus, a relatively small decrease in intra-cellular Ca2+–ATPase transport activities can accountfor the observed effects of oligomycin on Ca2+ releasefrom ICaS in spermatogenic cells.

Changes in adenine nucleotide pools and the subse-quent regulation of transport ATPases in spermatoge-nic cells can be a possible mechanism linking changesof mitochondrial oxidative phosphorylation andchanges in [Ca2+]i in spermatogenic cells. One aspectthat can be modified by changes in adenine nucleotideconcentrations was the phosphorylation–dephosphory-lation state of transport systems involved in intracellu-lar Ca2+ homeostasis. To explore the possibleinvolvement of protein phosphatases connectingchanges in adenine nucleotide pools and Ca2+ homeo-stasis in round spermatids, we used okadaic acid, aninhibitor of type 1 and 2A protein phosphatases. Prein-cubation of the cells (3 min) with okadaic acid (2 µM)did not change significantly either basal [Ca2+]i inround spermatids in suspension, or the subsequentrate of rise in [Ca2+]i induced by oligomycin in thesecells (n = 3). Thus, type 1 and 2A protein phosphatasesdo not appear to play a major role in basal intracellular

Ca2+ homeostatic mechanisms, or in the mechanismsresponsible for the rapid response of intracellular Ca2+

to inhibition of mitochondrial metabolism.

3.2. Effects of mitochondrial energymetabolism on pHi in rat pachytenespermatocytes and round spermatids

In the absence of external substrates (30 min at 33 ºCin a 95% O2/5% CO2 atmosphere), round spermatidsand pachytene spermatocytes have pHi values of7.1 ± 0.1 (n = 11) and 7.0 ± 0.1 (n = 9) respectively. Ad-dition of lactate induced a rise in pHi in these cells.Subsequent addition of oligomycin (figure 2A), antimy-cin (not shown), or DCCD (not shown), induced arapid decline in pHi.

Since the regulation of pHi in spermatogenic cellswas mainly performed by V-type H+–ATPases (Osseset al., 1997), we decided to explore if the inhibition ofV-type H+–ATPase could explain the intracellularacidification induced by the inhibition of oxidativephosphorylation in round spermatids. For that pur-pose, we obtained the dose–response curve for bafA-induced initial rate of intracellular acidification. Thisdose–response curve did not show saturation charac-teristics up to 8 µM bafA (figure 2B). Oligomycin in-duced an initial rate of pHi acidification that wasequivalent to the effect of approximately 8 µM bafA.The non-saturating bafA dose–response curve sug-gested to us that this compound was affecting otheraspects of acid-base metabolism besides V typeH+–ATPase. Thus, we tested the effect of bafA onmitochondrial oxidative phosphorylation. At concen-trations ≥ 4 µM, bafA induced an increase of QO2 inround spermatids, both in the presence or absence ofoligomycin. In the presence of oligomycin, CCCP (anuncoupler of oxidative phosphorylation) was unable tofurther stimulate QO2 after bafA addition (not shown).These data strongly suggest that bafA, besides beingan inhibitor of V-type H+–ATPases, was also affectingmitochondrial function in round spermatids, acting asan uncoupler. Thus, because of this bafA effect onmitochondrial oxidative phosphorylation, we couldnot determine if the oligomycin effect of inducingintracellular acidification was related to a partial ortotal inhibition of a plasma membrane H+–ATPase.

3.3. Relation between [Ca2+]i and pHichanges in round spermatids

Changes in oxidative phosphorylation induced si-multaneous changes in [Ca2+]i and pHi in spermatoge-nic cells. In order to test if these changes depended oneach other, we induced changes in [Ca2+]i and pHi byreleasing Ca2+ from intracellular stores using tsg, or byadding weak acids or bases, respectively. Thus, in-creases in [Ca2+]i induced by addition of tsg (100 nM)



did not produce significant changes in pHi in roundspermatids (n = 3, not shown). Conversely, a decreasein pHi from 7.3 to 7.0, induced by addition of 15 mMisobutyric acid to round spermatids, produced astepped change in [Ca2+]i of approximately 10 ± 6 nM(n = 3) in the absence of external Ca2+. Comparatively,

a similar change in pHi was produced after approxi-mately 200 s of oligomycin addition to the cells, a timelapse at which oligomycin induced a rise in intracellu-lar Ca2+ of approximately 300 nM. Furthermore, addi-tion of oligomycin followed by 5 mM NH4Cl 5 s later,increased the pHi to approximately 7.7 and induced arate of [Ca2+]i increase that was not different from therate of [Ca2+]i increase induced by oligomycin alone(n = 3). These data strongly suggest that the [Ca2+]i andpHi changes induced by changes in mitochondrialmetabolism do not depend on each other but aretriggered by a common mechanism.

3.4. Effect of glucose metabolism on [Ca2+]iand pHi in rat spermatogenic cells

It is known that the metabolism of glucose cannotmaintain high levels of intracellular ATP in rat roundspermatids (Nakamura et al., 1982). In fact, glucosemetabolism induced a net ATP hydrolysis in sperma-tids without changes in mitochondrial QO2, apparentlyby activation of a substrate cycle catalysed by phos-phofructokinase and fructose–1,6–bisphosphatase (Na-kamura et al., 1982; Grootegoed et al., 1986; Reyes etal., 1990). Thus, the impact of glucose metabolism on[Ca2+]i and pHi homeostasis in spermatogenic cellswas interesting to explore, because glucose can be ametabolite linking Sertoli and spermatogenic cell func-tion, and also because glucose metabolism produces achange in adenine nucleotide pools in round sperma-tids without inhibiting mitochondrial activity, allowingto test if the changes induced by glucose on [Ca2+]i andpHi were in agreement with their expected changes inintracellular adenine nucleotides concentrations.

Thus, we tested the effects that a 1 h incubation inKH medium supplemented with 10 mM glucose hadon [Ca2+]i and pHi in rat pachytene spermatocytes andspermatids. The results, displayed in table I show thatin rat spermatids the incubation with glucose pro-duced a marked rise in [Ca2+]i and a decrease in pHi.Pachytene spermatocytes did not show a significantincrease in [Ca2+]i when incubated with 10 mM glu-cose. Similarly, the pHi of pachytene spermatocytesshowed a non-significant lower value in 10 mM glu-cose as compared to lactate-containing media whenanalysed by ANOVA followed by Bonferroni test (tableI). However, in paired experiments, the pHi decrease inpachytene spermatocytes in glucose-containing mediaas compared to lactate-containing media was signifi-cantly different (p < 0.05, n = 3). Besides demonstratingthat glucose metabolism can modify [Ca2+]i and pHihomeostasis in spermatogenic cells, these data alsosuggest that changes in adenine nucleotide pools aresufficient to produce significant [Ca2+]i changes inround spermatids. These changes in adenine nucle-otides, together with a metabolic acid load, are likely to

Figure 2. A: Intracellular pH of rat pachytene spermatocytes(open circles) and round spermatids (closed circles) determinedusing BCECF-loaded cells. The cells were previously incubated at33 ºC in the absence of extracellular energy substrates for20 min in a 95% air/5% CO2 atmosphere. At the indicated times1 mM L-lactate (pH 7.4) or 5 µM oligomycin was added to the cellsuspension. The data shown are representative of measurementsperformed in four different cell preparations with similar results.B: Dose–response curves for the effect of bafilomycin A onintracellular pH acidification in round spermatids loaded withBCECF. The initial rate of intracellular acidification was estimatedfrom a linear regression of the data between 5 and 60 s afterbafilomycin A addition to the cell suspension in the presence of5 mM L-lactate. The dashed line represents a second-degreepolynomial fit of the data and was drawn for reference purposes.



be mediating the changes in pHi of round spermatidsand pachytene spermatocytes.

3.5. Effects of mitochondrial inhibitorson intracellular adenine-nucleotide levels inrat spermatogenic cells

As shown above, our data pointed toward changesin adenine nucleotide concentrations as a likely mecha-nism linking mitochondrial metabolism and intracellu-lar Ca2+ and pH homeostasis in spermatogenic cells. Tocorrelate if the changes in [Ca2+]i and pHi induced byinhibitors of mitochondrial oxidative metabolism werekinetically related to changes in cellular nucleotides,we determined the time course of intracellular ATP,ADP and AMP in rat spermatids after inhibition ofoxidative phosphorylation (figures 3A, B and C). Theinitial ATP, ADP and AMP concentrations in rat sper-matids were 79 ± 18, 11 ± 3 and 7 ± 3 nmoles·mg–1

protein, respectively (mean ± SEM of duplicatemeasurements performed in six different cell prepara-tions). Considering that rat round spermatids haveapproximately 6 µL·mg–1 protein (Reyes et al., 1990),the average intracellular concentrations of ATP, ADPand AMP would be approximately 13, 2, and 1 mM,respectively. The time course of cell ATP decrease wasfitted to a single exponential decay curve (timeconstant = 6.1 min), corresponding to the kinetics ofirreversible exit of a compound from a single chemicalcompartment. The time course of cell ADP content wasfitted to a curve corresponding to the derivation to-ward a steady-state concentration of a intermediaryproduct. The time course of the calculated ATP/ADPratio (single exponential) is shown in the insert of figure3B (time constant = 2.0 min). The rise of cell AMP wasapproximately linear.

Similar net effects of inhibitors of oxidative phos-phorylation were observed on cell adenine nucleotidesin pachytene spermatocytes. In these cells, the basalATP, ADP and AMP contents were 88 ± 14, 8 ± 2 and10 ± 2 nmoles·mg–1 protein, respectively (n = 3). Anti-mycin or oligomycin produced a drop of 51 ± 6%

(n = 3) of the intracellular levels of ATP after 3 min ofexposure to the inhibitors.

Because with external lactate as substrate the ATPsynthesised by round spermatids was provided almostentirely by the mitochondria (Reyes et al., 1990), uponinhibition of mitochondrial ATP production, the initialrate of ATP decrease (or ADP increase) reflects the rateof ATP utilisation by these cells. From the rate constantof decrease in intracellular ATP and the initial ATPcontent of the cells, we estimated that in conditions ofinhibited ATP production, rat round spermatids wereutilising ATP at a rate of 15.2 nmoles·min–1·mg–1 pro-tein. A similar calculation was made from the initialrise in intracellular ADP, giving an ATP consumptionof 16.0 nmoles·min–1·mg–1 protein, in good agreementwith the number estimated from the decrease inATP content. The total adenine nucleotide(ATP + ADP + AMP) content decreased approximately20% after 10 min of inhibition of mitochondrial oxida-tive metabolism in rat round spermatids (insert figure3C). This decrease was likely due to purine nucleotidecatabolism, since a peak with a retention time similarto adenosine appeared in the chromatogram (notshown).

3.6. Uptake of Ca2+ by rat spermatogeniccell mitochondria

In our previous work (Berrios et al.,1998) we showedthat in cells metabolising lactate, round spermatidmitochondria were able to store a small amount ofCa2+ (approximately 20 nM) under basal [Ca2+]i. Inthese cells, oligomycin and antimycin (or rotenone)produced a similar inhibition of oxygen consumption.However, antimycin or rotenone induced a larger risein [Ca2+]i than did oligomycin (significantly different,n = 6, p < 0.01, paired test). This effect is well evidencedin figure 4, where a steady [Ca2+]i of approximately150 nM was reached after treatment of round sperma-tids with oligomycin. Antimycin induced a rise in[Ca2+]i of about 200 nM over and above the levelsinduced by oligomycin. In average, rotenone or anti-

Table I. Intracellular [Ca2+] and pH in pachytene spermatocytes and round spermatids

[Ca2+]i (nM) pHi (units)

KH–lactate KH–glucose KH–lactate KH–glucose

pachytene spermatocytes 42 ± 6 39 ± 6 7.26 ± 0.15 6.92 ± 0.08round spermatids 36 ± 3 93 ± 6* 7.43 ± 0.27 6.47 ± 0.13¥

Triplicate measurements were performed in three different cell preparations after 1 h of incubation at 33 ºC under a 95% air/5% CO2atmosphere.* Significantly different from pachytene spermatocytes in KH–lactate and KH–glucose, and round spermatids in KH–lactate (p < 0.05,Bonferroni t test)¥ Significantly different from pachytene spermatocytes in KH–lactate and round spermatids in KH–lactate (p < 0.05, Bonferroni t test)



mycin induced a rise of 180 ± 80 nM (SD, n = 5) in[Ca2+]i above the levels reached after oligomycin treat-ment of round spermatids. Similar results were ob-served in pachytene spermatocytes (not shown). It isworth noting that tsg was unable to release more Ca2+

after oligomycin treatment (see above), indicating thatthe Ca2+ released by antimycin or rotenone subsequentto oligomycin was not originating from ICaS. Oligomy-cin inhibits the H+ flow through the mitochondrial ATPsynthase and is expected to maintain the H+ gradientand the electrical potential across the inner mitochon-drial membrane, i.e., the forces involved in the electro-phoretic uptake of Ca2+. In contrast, antimycin orrotenone, by inhibiting the electron flow in the mito-chondria electron chain, is expected to cause a collapseof the H+ gradient and electrical potential (Scarpa,1979). Thus, our results suggest that in rat spermatids

the mitochondria took up Ca2+ when their membraneswere energised in the presence of oligomycin and[Ca2+]i was raised above the basal concentration.

3.7. Vital location of rat spermatogenic cellmitochondria and endoplasmic reticulum

[Ca2+]i and pHi respond almost immediately to theinhibition of mitochondrial oxidative phosphorylationand to the addition of lactate (figures 1 and 2, A and B).These facts suggest that a close association, functionalor anatomical, exists between spermatogenic cell mito-chondria and the transport systems that regulate[Ca2+]i and pHi in spermatogenic cells. It has beenreported that, in fixed cells, spermatid mitochondriaoccupy a location adjacent to the plasma membrane, incontrast to the mitochondria of pachytene spermato-

Figure 3. A. Relative intracellular levels of ATP in rat roundspermatids. At zero time, antimycin (5 µM) was added to the cellsuspension. Basal levels of ATP in rat spermatids were79 ± 18 nmoles·mg–1 protein. B. Relative intracellular levels ofADP of rat round spermatids. At zero time, antimycin (5 µM) wasadded to the cell suspension. Basal levels of ADP in rat sperma-tids were 11 ± 3 nmoles·mg–1 protein. The insert shows the timecourse of the calculated ATP/ADP ratio in each nucleotide deter-mination. C. Relative intracellular levels of AMP of rat roundspermatids. At zero time, antimycin (5 µM) was added to the cellsuspension. Basal levels of AMP of rat spermatids were7 ± 3 nmoles·mg–1 protein. Insert: Total adenine nucleotide con-tent (ATP + ADP + AMP) of rat round spermatids.



cytes which are distributed more randomly in theircytoplasm (De Martino et al., 1979; Romrell et al.,1976). We used the mitochondrial fluorescent probeMitoTracker Red CM-H2Xros that stains metabolicallyactive mitochondria, to visualise vitally these or-ganelles in isolated pachytene spermatocytes andround spermatids. Figure 5A show that round sperma-tid mitochondria were located mainly adjacent to theplasma membrane, but also some of these organelleswere observed in the cytoplasm and adjacent to thenucleus. In contrast, pachytene spermatocyte mito-chondria were distributed in the cytoplasm, formingclusters of these organelles. The ER of spermatogeniccells (figures 5B and C) showed a distribution notnecessarily correlated with the differential distributionof their mitochondria. Thus, in pachytene spermato-cytes ER-Tracker showed a distribution in the cyto-plasm and surrounding the nucleus, and also thelabelling of a prominent organelle, identified as theGolgi system by labelling the cells with BODIPYtTRceramide (not shown). In round spermatids the distri-bution of ER-Tracker was similar to pachytene sperma-tocytes with a stronger labelling of the perinuclearregion, but with no preferential distribution in associa-tion with metabolically active mitochondria.

4. DISCUSSION

Rat spermatids (Berrios et al., 1998; Osses et al.,1997) and also pachytene spermatocytes (Reyes et al.,unpublished results) rely mainly on Ca2+-Mg2+ AT-

Pases and V-type H+–ATPases for their intracellularhomeostasis of Ca2+ and pH, respectively. The proper-ties of these [Ca2+]i and pHi regulatory transport sys-tems and the metabolic characteristics of meiotic andpostmeiotic spermatogenic cells, make [Ca2+]i and pHiin these cells especially susceptible to be modulated bythe energy state and metabolic fluxes. In agreement,our measurements of [Ca2+]i and pHi in pachytenespermatocytes and round spermatids showed that thechanges in mitochondrial metabolism were rapidlytranslated into changes of [Ca2+]i and pHi homeostasisin the absence or presence of external Ca2+. Approxi-mately 15 s after inhibiting mitochondrial oxidativephosphorylation, [Ca2+]i and pHi homeostasis wereimpaired. The Ca2+ release induced by oligomycin wasnot provided by the mitochondria, since these or-ganelles were taking up Ca2+ under this condition.Besides, glucose, which does not inhibit mitochondrialmetabolism, can cause an increase of [Ca2+]i in roundspermatids. Because saturating concentrations of tsginduced the release of only a small amount of Ca2+

after oligomycin addition, we can conclude that theCa2+ released to the cytosol by the action of oligomycinoriginated from tsg-sensitive intracellular Ca2+ stores.

The effects of glucose on [Ca2+]i in round spermatidsand pachytene spermatocytes correlated with its effectson adenine nucleotide pools (Grootegoed et al., 1986;Nakamura et al., 1982; Reyes et al., 1990), pointingtoward changes in adenine nucleotides as the neces-sary link between mitochondrial metabolism and[Ca2+]i homeostasis in spermatogenic cells. Consistentwith this notion, the inhibition of oxidative phospho-rylation induced a decrease in intracellular ATP levels(and the related changes in intracellular ADP and AMPconcentrations). However, [Ca2+]i and pHi respondedto inhibition of mitochondrial metabolism when theaverage levels of intracellular ATP were relatively high(> 5 mM) and more than 10 times the reported Kmvalues for ATP in Ca2+ and H+–ATPases (Moriyamaand Nelson, 1987; Inesi et al., 1992). Soltoff and Mandel(1984), when studying the ATP dependence of Na+–K+

ATPase-mediated transport in renal proximal tubulesreported a similar result. This tight coupling betweenmitochondrial function and active transport that can-not be accounted for only by a decrease in the avail-ability of ATP as substrate, can be postulated to arisefrom two sources. First, mitochondrial ATP productionin these cells can be localised to the sites where ATPwas being utilised by the transport ATPases, i.e., trans-port ATPases sense the local change in nucleotideconcentrations before the changes in average cellnucleotides take place. Second, Ca2+– and H+–ATPasesinvolved in [Ca2+]i and pHi homeostasis could beregulated (allosterically or through phosphorylation)by intracellular nucleotides. These two possibilities arenot mutually exclusive.

Figure 4. Intracellular Ca2+ of rat round spermatids in suspen-sion incubated with 10 mM L-lactate. At the indicated times,oligomycin (5 µM) and antimycin (5 µM) were added to the cellsuspension. The data showed are representative of measure-ments made in four different cell preparations with similar results.



Our data of vital localisation of metabolically activemitochondria and ER in spermatogenic cells suggesteda cellular anatomical relation between mitochondriaand the sites of ATP utilisation for plasma membrane(PM) H+ and Ca2+ transport in round spermatids.Similarly, a microanatomical correlation existed be-tween metabolically active mitochondria and ER (en-doplasmic reticulum Ca2+ transport) in pachytene

spermatocytes. However, no evident cellular anatomi-cal correlation was shown between metabolically ac-tive mitochondria and the sites of PM H+ transport inpachytene spermatocyte or for ER Ca2+ transport inround spermatids. The absence of a microanatomicalcorrelation between metabolically active mitochondriaand H+ transport in pachytene spermatocytes or Ca2+

transport in round spermatids prevents it to be pro-

Figure 5. A. Composite image of differential interference contrast and fluorescence confocal images of round spermatids and pachytenespermatocytes labelled with MitoTracker Red CM-H2Xros to show the metabolically active mitochondria. The images were taken with a100 × oil immersion objective. The optical sections in this microphotograph were approximately 1 µm. The bar represents 10 µm. B,C.Fluorescence images of ER-Tracker distribution in round spermatids (B) and pachytene spermatocytes (C). The images were taken with a100 × oil immersion objective and corrected for out-of-focus light as described in the text. The bar represents 10 µm.



posed as a general mechanism. However, we cannotdiscard that it may be a mechanism that kineticallylinks changes in the rate of ATP production with ATPutilisation, in H+ transport systems in round sperma-tids or in ICaS Ca2+ transport systems in pachytenespermatocytes. Whether through a topological associa-tion between ATP-producing and ATP-consuming sitesin some cases, changes in cell nucleotides appear toconnect metabolic energy pathways and ion transportATPases in these cells. Our data also suggest that inthese cells intracellular nucleotides played a regulatoryrole in H+– and Ca2+–ATPase activity. Although aregulatory role for serine/threonine phosphorylationhave been described in transport ATPases, the absenceof an effect of okadaic acid suggest that dephosphory-lation by PP1/PP2A protein phosphatases was not themain factor responsible for the fast coupling betweenmitochondrial metabolism and Ca2+ transport activity.The nucleotide parameter that seems to respond asrapidly as the changes in Ca2+ homeostasis is theATP/ADP ratio (see insert figure 2B, time constant = 2.0min). In fact, ATP and ADP have been described asallosteric regulatory nucleotides for V-typeH+–transport ATPases (Moriyama and Nelson, 1987).An allosteric regulation by adenine nucleotides hasbeen controversial for ER Ca2+–transport ATPases (e.g.,Inesi et al., 1992). However, ADP can bind to thenucleotide-binding site in this enzyme, competing forthe binding of ATP. Thus, our analysis strongly suggestthat a regulation by adenine nucleotides, specificallyby the ATP/ADP, can provided the link between mito-chondrial metabolism and transport ATPases in roundspermatids and pachytene spermatocytes.

It is highly likely that the linkage between mitochon-drial metabolism and pHi changes was also providedby changes in adenine nucleotide pools. However,because bafA uncoupled the oxidative phosphorylationin round spermatids at concentrations ≥ 4 µM, wecould not estimate if the effect of oligomycin on pHicould be accounted for by a partial or total inhibition ofV-type H+–ATPases. Similarly, because of this bafAeffect on oxidative phosphorylation, we cannot ruleout that part of the intracellular acidification effect ofmitochondrial inhibitors could be due in part to ametabolic acid load.

4.1. Can glucose be a regulatory substratein postmeiotic spermatogenic cells?

It is known that glucose metabolism in round sper-matids can decrease the energy status of these cells(Grootegoed et al., 1986; Nakamura et al., 1982; Reyeset al., 1990). Accordingly, we found that incubation ofround spermatids in KH containing 10 mM glucoseproduced a marked rise in [Ca2+]i and a drop in pHi. Inpachytene spermatocytes, this effect of glucose was notobserved for [Ca2+]i, and was less pronounced but

significant for pHi (table I). In pachytene spermato-cytes, glucose did not decrease significantly intracellu-lar adenine nucleotides, and its effects on pHi could beattributed, in principle, to a metabolic acid load due toglucose metabolism. Our data can explain, in terms ofglucose effects on adenine nucleotide levels, the resultsof a higher basal [Ca2+]i in round spermatids as com-pared to pachytene spermatocytes maintained inglucose-containing media reported by Treviño et al.(1998). Similarly, low intracellular pH has been re-ported for both pachytene spermatocytes and roundspermatids by Santi et al.(1998) in a glucose-containingmedia (see Osses et al., 1997). Our results suggest thatthis low pHi is likely due to a low energy charge inround spermatids and a metabolic acid load inpachytene spermatocytes. Thus, glucose can differen-tially modulate [Ca2+]i and pHi in meiotic and postmei-otic spermatogenic cells, strongly suggesting that it canbe part of the network of signals that connect Sertoliand spermatogenic cells in the seminiferous tubule.

In conclusion, rat pachytene spermatocyte andround spermatid intracellular Ca2+ and H+ homeosta-sis are highly sensitive to changes in oxidative metabo-lism and availability of substrates such as glucose andlactate. Our data strongly suggest that the mechanismby which the changes in glycolytic and oxidative me-tabolism are translated into changes in [Ca2+]i and pHiin these cells involved nucleotide regulation of trans-port ATPases. Because energy substrates used by sper-matogenic cells are expected to be provided by Sertolicells in a hormonally regulated manner, [Ca2+]i andpHi of meiotic and postmeiotic spermatogenic cells areexpected to be regulated by the hormone-dependentglucose transference and lactate production of Sertolicells. Thus, our results provide further arguments for ahormonally regulated metabolic link between Sertoliand spermatogenic cells that involves [Ca2+]i and pHiin spermatogenic cells. The ability of glucose to affectdifferentially the energy status, [Ca2+]i and pHi ofspermatogenic cells at different stages of development,suggests that this molecule is part of the hormonallyregulated signals connecting Sertoli and spermatogeniccells in the seminiferous tubules.

To the best of our knowledge, our results providesupport for the first comprehensive mechanisms so farproposed that links Sertoli cell activity with intracellu-lar signaling in meiotic and postmeiotic spermatogeniccells. Thus, as a working hypothesis we would like topropose that intracellular Ca2+ and pH of spermatoge-nic cells in the seminiferous tubules are changing withthe FSH-regulated glycolytic activity of Sertoli cells(lactate and glucose provision toward the adluminalcompartment). The magnitude of the [Ca2+]i and pHichanges are expected to differ in pachytene spermato-cytes and spermatids, controlling events like meioticand spermiogenic progression, respectively.



Acknowledgments. Our appreciation to Dr. J. Hidalgofor his help with image processing. This work wassupported by Grants from FONDECYT 1990689 andDGIP/UCV.

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energy metabolism and its linkage to intracellular ca2+ and ph regulation in rat spermatogenic cells

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