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a Departments of Internal Medicine and
b Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9135
c Departments of Animal Science and
d Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4458
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data established the following: 1) L-CPT I was the sole isoform detected in immature testis. 2) Expression of the M-CPT I gene was associated only with meiotic and postmeiotic germ cells. 3) Adult testis contains a mixture of the L- and M-CPT I enzymes, the L and M form dominating in extratubular cells and spermatids, respectively. Mature epididymal spermatozoa appear to be devoid of CPT I activity while possessing abundant levels of CPT II and CAT. 4) Five days of dietary etomoxir treatment at a dose that resulted in essentially complete inhibition of CPT I in liver, heart, skeletal muscle, and kidney was totally without effect on either the L- or M-type enzyme in the testis of mature rats.
The data point to an important role for transient expression of M-CPT I, coupled with sustained activity of CAT, in the maturation and/or function of rat sperm. They also suggest that, at least in the case of one CPT I inhibitor (etomoxir), the testis is unusually resistant to the agent when given orally.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has recently come to light that the L and M forms of CPT I are expressed in a variety of tissues other than those for which they are named, and in variable and unpredictable amounts [2–5]. In the course of one of those studies [5], we were struck by the fact that in both the rat and mouse, whole testis is extremely rich in the mRNA for M-CPT I, suggesting a role for this enzyme in reproductive function. In the present study, we have been able to expand upon that initial observation by defining at which developmental stage and in which cell types the M-CPT I gene is turned on in the testis. The data have important implications regarding fuel utilization in the developing sperm and raise the possibility that fatty acid oxidation plays a hitherto unrecognized and essential role in the maturing male germ cell. We have also begun to address the issue of possible undesirable side effects of CPT I inhibitors on sperm development should the use of such compounds ever be contemplated in the pharmacological management of diabetes [1, 6].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature male Sprague-Dawley rats were fed a standard laboratory chow (4% fat w:w) ad libitum. When used for sperm isolation they weighed > 400 g, but for all other studies body weights were 275–300 g. Immature, suckling rats (~28 g) were withdrawn from the dam at 14 days of age. All animals were housed with lighting from 1000–2200 h and were used for experiments between 1000–1200 h. Tissues were obtained from mature rats anesthetized with sodium pentobarbital and from immature animals killed under methoxyflurane anesthesia. All animal studies were performed in accordance with the Guiding Principles for the Care and Use of Research Animals of the Society for the Study of Reproduction.
Preparation of Mitochondria
Mitochondria from liver, heart, kidney, and hind limb skeletal muscle of mature rats were prepared as described previously [7] and resuspended in 150 mM KCl/5 mM Tris-HCl, pH 7.2 (buffer A). Whole-testis mitochondria from mature and immature animals were obtained as follows: after removing the tunica albuginea and visible blood vessels, ~0.14–0.44 g (immature) to ~1 g (mature) of tissue was homogenized on ice in 10–15 volumes of buffer composed of 250 mM sucrose/5 mM Tris-HCl, pH 7.2 (5 sec at setting 10, large probe, Tissumizer polytron; Tekmar Co., Cincinnati, OH), and then processed as described above for liver and muscle preparations.
Preparation of Testis Cell Fractions
Separation of testis cell fractions was achieved through a velocity sedimentation procedure similar to that described by Meistrich [8]. Testes (~2 g each) from an anesthetized mature rat and stripped of the tunica albuginea and visible blood vessels were digested at 33°C in 50 ml of germ-cell Minimal Essential Medium (GC-MEM: 10 mM HEPES, 6 mM sodium-lactate, 1 mM sodium-pyruvate, and 2 mM glutamine in MEM with Earle's salts [pH 7.3] plus collagenase [0.6 mg/ml]). Seminiferous tubules were dispersed by shaking (100 cycles/min, 15 min, 33°C) and allowed to settle. The supernatant was combined with those from subsequent 25-ml GC-MEM washes (stored on ice) and designated "extratubular cells." Further digestion (130 cycles/min, 15 min, 33°C) in 50 ml of GC-MEM plus trypsin (0.5 mg/ml) and DNase I (1 µg/ml) was stopped by addition of soybean trypsin inhibitor (SBTI, 0.5 mg/ml). Preparations were filtered through a 70-µm nylon mesh and centrifuged (450 x g, 10 min, 4°C). The germ cell pellet was washed (25 ml ice-cold GC-MEM with 5 mg/ml BSA, 0.25 mg/ml SBTI, 0.5 µg/ml DNase I), recentrifuged, and resuspended in 24 ml of cold wash buffer.
Cell separation was carried out at 4°C by employing a Sta-Put cell separator apparatus (ProScience Inc., Toronto, Canada) and using a protocol patterned after the equipment instructions. Briefly, 60 ml of GC-MEM was loaded and allowed to flow toward the sedimentation chamber (10 ml/min). Subsequently, the germ cell preparation was loaded, followed by a rinse (10 ml GC-MEM) when the chamber was almost emptied of cells. A BSA gradient was formed in the reservoir by initiating maximal flow over ~20 min from a two-chamber gradient maker upstream from the loading chamber (2% or 4% BSA in GC-MEM in the proximal and distal chambers, respectively) and cells were allowed to settle for 1.5 h. About 90 fractions (11 ml each) were collected and centrifuged (450 x g, 10 min, 4°C), and the cell pellets were examined microscopically for germ cell identification and enrichment. Spermatid-enriched fractions (generally numbers 35–70) contained round cells, ~10 µm in diameter, with a subpopulation possessing flagella at differing levels of development [8]. Relative quantification of each spermatid subpopulation was not performed. These were combined and centrifuged (1200 x g, 10 min, 4°C), as were the extratubular cells from above. For CPT assays (see below), cells were washed twice in 5–10 ml ice-cold PBS, resuspended in 2 ml of buffer A, and homogenized on ice using 5 strokes of a tight-fitting glass homogenizer. After centrifugation (16 000 x g, 10 min, 4°C), the pellets were resuspended in 1 ml (spermatids) or 2 ml (extratubular cells) of buffer A.
Preparation of Spermatozoa
Spermatozoa were isolated by placing ~1.5 cm of the cauda epididymidis into a 100-mm Petri dish containing 20 ml of warmed (37°C) Dulbecco's Modified Eagle's Medium. Several incisions were made along the epididymis, allowing mature sperm to be released and accumulate in the surrounding medium over a period of 30 min. After tissue fragments were removed, a sperm pellet was obtained by centrifugation (1200 x g, 10 min, 4°C). This was washed twice with 10 ml of ice-cold PBS and resuspended in 3 ml of ice-cold hypotonic potassium phosphate buffer (5 mM, pH 7.0), a treatment that removes the cell membrane from mitochondria-rich sperm tails but leaves the mitochondria intact and metabolically sound [9]. However, this type of preparation failed to display significant CPT I activity. An alternative approach, using conventional Polytron homogenization of the sperm cells in a manner similar to that used for other sample types, was similarly negative. In an attempt to facilitate measurement of CPT I in spermatozoa, a membrane fraction was prepared from the hypotonically treated spermatozoa and extracted with mild detergent under conditions known to enrich for CPT I [10]. In this procedure, sperm were freeze-thawed three times and then centrifuged at 14 500 x g for 10 min at 4°C. The membrane pellet was resuspended in the original volume (3 ml) of potassium phosphate buffer (5 mM; pH 7.0), made 1% (v:v) with Tween-20 and kept on ice for 30 min, mixing frequently. Tween-20 selectively extracts CPT II while leaving CPT I intact in the membrane [10]. Further centrifugation yielded a membrane pellet that should have contained the CPT I activity if any was present.
CPT Assay
With preparations containing intact mitochondria, measurement of CPT I was performed as described by Esser et al. [11] in an assay mixture containing 500 µM [14C]-L-carnitine and 50 µM palmitoyl-CoA with the indicated concentrations of malonyl-CoA. CPT activity remaining in the presence of 100 µM malonyl-CoA (generally 10–15%) was considered to represent malonyl-CoA-insensitive CPT II exposed during mitochondrial preparation. To normalize for variability in this parameter across tissues/preparations, CPT I activities reported herein are values corrected for the CPT II contribution and thus represent the malonyl-CoA-sensitive enzyme [11]. For assay of CPT II, preparations were made 1% with regard to octylglucoside and kept on ice for 30 min before assay, with frequent mixing. This detergent solubilization procedure inactivates CPT I and releases CPT II in active form [10]. Assay times were generally 4–8 min.
Protein concentration was assayed by the method of Lowry et al. [12].
Etomoxir Experiments
[3H]Etomoxir labeling of CPT I isoforms in mitochondria from adult rat liver and heart, and from mature and immature testes, was essentially as described by Brown et al. [4] except that the "activation mixture" included 100 mU acyl-CoA synthetase (Boehringer Mannheim, Indianapolis, IN) and 10 µM [3H]etomoxir [2]. Radiolabeled membrane fractions were subjected to SDS-PAGE and fluorography [4].
To assess the impact of dietary etomoxir upon CPT I activity in the testis, mature rats were fed powdered normal rat chow ad libitum with or without a racemic mixture of sodium-etomoxir (Byk Gulden Pharmaceuticals, Konstanz, Germany) at 0.05% (w:w). This diet was introduced at ~1200 h on Day 0 and continued until the morning of Day 5. At that time, extratubular cells and spermatids were isolated from the testes for assay of CPT I and CPT II as described above. For comparison, the level of each enzyme was also measured in mitochondria prepared from liver, heart, skeletal muscle, and whole kidney.
Northern Blot Analyses
The abundance of mRNA for L-CPT I and M-CPT I was determined in testis obtained from mature and immature rats. Single testes from the former were rinsed in cold PBS and placed in liquid N2 after removal of the tunica albuginea and visible blood vessels. Testes from 7 immature pups (pooled into 4–5 testes/preparation) were treated similarly except that the tunica was not removed before storage. Total RNA was obtained by employing Trizol (Life Technologies, Grand Island, NY), and 20 µg was analyzed using formaldehyde gels [13]. Hybridization was carried out for 1.5 h at 65°C in a hybridization oven (Biometra, Gottingen, Germany) according to the instructions for the Rapid-Hyb buffer system (Amersham, Arlington Heights, IL). Nylon membranes were washed three times (65°C, 15 min) in 0.2-strength SSC (single-strength SSC = 0.15 M sodium chloride, 0.015 M sodium citrate)/0.2% SDS. Double-stranded DNA probes were generated through polymerase chain reaction (PCR) amplification of isoform-specific cDNA fragments from plasmid templates containing coding regions of rat L- or M-CPT I. Probes corresponded to positions +1 to +995 and +464 to + 1879 of the rat L- and M-CPT I cDNAs, respectively [14, 15], and were radiolabeled with [
-32P]dCTP by random hexamer methods [13].
In Situ Hybridization
Construction of pBluescript plasmids containing cDNAs for rat L-CPT I and M-CPT I has been described [11, 15]. Detection of mRNAs on cross-sections of testis by in situ hybridization was performed according to Ing and Tornesi [16]. Antisense RNA probes labeled with [35S]UTP (New England Nuclear, Boston, MA) were transcribed in vitro from pBluescript templates linearized at SalI or BglII sites within the cDNAs. To estimate nonspecific hybridization on adjacent sections, sense probes were also synthesized. After hybridization and RNase treatment, slides were exposed to x-ray film (XAR-5; Kodak, New Haven, CT) for 2 days. They were then coated with autoradiographic emulsion (NTB; Kodak) and developed 2 wk later.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine which cell type(s) in the testis is responsible for expression of the M-CPT I gene, we used the technique of in situ hybridization in which slices of the organ were probed with a radioactive antisense mRNA for the muscle enzyme. As seen from Figure 3, the results were informative and unequivocal. The low-power autoradiograph shown in Figure 3a illustrates the strong and highly specific labeling of the seminiferous tubules (ST) of the adult animal. One long loop is particularly clear (large arrow). The specific reaction of the probe is confirmed in Figure 3b, in which the seminiferous epithelium (SE) of an adult rat has been treated with a "sense" probe and only a few silver grains are present. The grains indicating hybridization of the antisense probe in the higher-magnification bright- and darkfield images (Fig. 3, c and d, respectively) of the same section of adult seminiferous tubule demonstrate several points. First, there was a strong and specific interaction of the riboprobe with the seminiferous epithelium, where primary spermatocytes (PS) and spermatids (Sd) reside (between paired arrows pointing toward each other). Second, Leydig (LC) and other interstitial cells between tubules and the spermatogonia (Sg) and myoid cells located at the basal limits of the tubules are not labeled. Third, because of the oblique nature of the cross-section, a number of spermatogonia are exposed and seen to be unlabeled (open arrow). At even higher power (Fig. 3e), grains are clearly seen to be concentrated towards the luminal side of the epithelium (primary spermatocytes and spermatids), and few grains are observed in the spermatogonia (basal side). Figure 3f shows antisense probe hybridization in a brightfield image of a section of testis from an immature rat, where germ cells are few. Leydig and Sertoli cells (SC) are unlabeled. Thus, labeling with the M-CPT I probe was associated exclusively with germ cells. Furthermore, whereas expression of the M-CPT I gene was undetectable in spermatogonia, it was clearly actively transcribed in spermatocytes and remained so in the spermatids (spermatogonia, primary spermatocytes and spermatids represent sequential stages in spermatogenesis). Whether the spermatozoon continues to express the M-CPT I message during its epididymal transit and final maturation could not be determined with certainty because we had difficulty obtaining mRNA from fully mature spermatozoa isolated from the caudal region of the epididymis. The reason might be that at this final stage of sperm development, gene transcription has declined to a low level [17].
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Similar studies were conducted using an antisense probe for L-CPT I. However, the signals obtained were very weak, suggesting that the expression level of L-CPT I mRNA was too low to be detected by in situ hybridization (data not shown). The very different exposure times required to detect the M-CPT I and L-CPT I signals on Northern blots of testes (Fig. 1) are entirely consistent with this interpretation.
As alluded to above, the L and M forms of CPT I differ markedly in certain kinetic properties, notably the sensitivity of the two enzymes to malonyl-CoA and their Km for the substrate carnitine. In keeping with the findings shown in Figures 1–3, the malonyl-CoA sensitivity of CPT I in mitochondria from whole, immature testis displayed an IC50 value (concentration needed for 50% inhibition) identical with that for the liver enzyme (~7 µM under the conditions used here; Fig. 4A). As expected [3], the IC50 for the skeletal muscle enzyme was much lower (~0.1 µM in the current series). The intermediate value of ~2 µM found for mature testis is entirely consistent with the presence of a mixture of the L- and M-CPT I isoforms [2–4]. Preparations of pachytene spermatocytes displayed a sensitivity to malonyl-CoA similar to that of the spermatids (not shown). Although studied less extensively, it was evident that the Km for carnitine of CPT I in immature testis was similar to that we routinely observe for the enzyme in rat liver (~40 µM), while the value for mature testis was found to lie between 120 and 200 µM, consistent with the presence of a significant amount of the higher-Km M isoform (Km ~500 µM in rat skeletal muscle). Qualitatively, these findings were reminiscent of the situation with rat heart mitochondria, which also express a mixture of the two CPT I proteins [2–4].
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That the L and M forms of CPT I in whole, mature testis were associated with specific cell populations is seen from the data in Figure 4B, which show that after fractionation of the tissue, the high- and low-IC50 species of CPT I were carried by the extratubular cells and spermatids, respectively. Interestingly, using a variety of techniques to isolate mitochondria or membrane fractions from fully developed epididymal spermatozoa, we found CPT I activity to be essentially undetectable, despite readily measurable levels of CPT II (5–25 nmol/min per mg protein depending upon the several methods of cell homogenization employed; c.f. control values for other tissues in Fig. 5, below). It appears, therefore, that the high level of expression of M-CPT I is transient, beginning in the primary spermatocyte, but ceasing with complete maturation of the spermatozoon, at which time neither isoform of CPT I is present in measurable amounts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alternatively, the need for CPT I during spermatogenesis might have more to do with the generation of acetylcarnitine. Acetyl-CoA derived from the oxidation of long-chain fatty acids can react with carnitine under the influence of carnitine acetyltransferase (CAT) to yield acetylcarnitine. In this context, it is noteworthy 1) that CAT activity also develops during spermatogenesis, reaches high levels by the spermatid stage, and (unlike CPT I) is fully retained in spermatozoa ([24] and confirmed by us in experiments not shown); and 2) that during their transit through the epididymis, the maturing sperm encounter extremely high levels of carnitine in the surrounding fluid (~60 mM in the rat), from which they generate a large reservoir of acetylcarnitine, which is thought to provide a ready source of energy during their post-ejaculation activity [25]. If fatty acids, perhaps in addition to pyruvate, act as a supplier of acetyl-CoA for acetylcarnitine synthesis, the need for an active CPT I at the spermatid stage would be explained. In this scenario, once a sufficiently high concentration of acetylcarnitine has been generated during passage of the sperm through the epididymis, CPT I activity might no longer be necessary. On the other hand, CAT would still be required for the regeneration of acetyl-CoA from acetylcarnitine during the sperm's extra-epididymal activity. We consider further speculation to be unwarranted at this time.
A second and even more perplexing issue is why the developing rat sperm should express the muscle rather than the liver variant of CPT I. The same question can be asked about brown and white adipose tissue in species such as the rat, hamster, and human [5], but at present a simple explanation is not obvious. In fact, given the extremely high level of carnitine found in epididymal fluid, expression of the high-Km M form of CPT I could perhaps be more reasonably explained on teleological grounds for the developing sperm than in the cases of those other tissues. However, knowledge of the carnitine concentration within the developing germ cells is not currently available.
Finally, given the interest that has been expressed in the potential therapeutic use of CPT I inhibitors in diabetes [6], the question of whether testicular CPT I might be vulnerable to attack by such agents in vivo will have to be addressed. As a first step in this direction, we treated a group of mature rats for 5 days with etomoxir (included in their diet at 0.05% w:w, a dose that we had previously shown to inhibit CPT I in rat liver and skeletal muscle). At the end of this period, CPT I and CPT II measurements were made on mitochondria prepared from liver, heart, skeletal muscle, kidney, and testis. Remarkably, whereas CPT I activity in liver, heart, skeletal muscle, and kidney was completely abolished by this maneuver (Fig. 5), no significant effect was observed on whole testis mitochondria (not shown). Both the CPT I of testicular extratubular cells (the L form) and that of spermatids (the M variant) remained totally unaffected (Fig. 5). Not surprisingly [26], there was no suppression of CPT II activity by etomoxir at any site; on the contrary, the drug caused a significant increase in the hepatic level of CPT II. It thus appears that, at least over a 5-day period and at the drug dosage used here, the rat testis is a privileged site in terms of susceptibility of its CPT I isoforms to attack by etomoxir. Whether this stems from a low permeability of the tissue to the drug and/or from an inability of testicular cells in vivo to generate enough of the activated form of the inhibitor, etomoxir-CoA, is not known. (It should be noted that the covalent [3H]etomoxir labeling of testicular CPT I shown in Figure 2 was achieved by direct exposure of mitochondria to the inhibitor in the presence of exogenous ATP, CoA, and acyl-CoA synthetase.) It seems unlikely that the apparent protection of testis CPT I from etomoxir in vivo relates to the blood-testis barrier in light of the lack of effect of the inhibitor on both extratubular and sperm cell populations that lie on opposite sides of this boundary [27]. Naturally, it remains a possibility that a much higher dose of the inhibitor would affect testicular CPT I.
In conclusion, despite the strong evidence for a crucial role for carnitine in sperm maturation, surprisingly little attention has been paid to the role of fatty acid ß-oxidation in this process. Whether the robust appearance and subsequent apparent disappearance of M-CPT I during rat sperm differentiation also applies to humans is unknown. However, the data presented here provide strong circumstantial evidence that fatty acid oxidation may be of greater importance in sperm development than has previously been thought. If so, a sperm-targetable and reversible inhibitor of CPT I could have interesting pharmacological potential. By the same token, it would seem prudent that studies similar to those conducted here with etomoxir be carried out with any other CPT I inhibitor that might be considered in the treatment of disease processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: J. Denis McGarry, U.T. Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235–9135. FAX: 214 648 2843; dmcgar{at}mednet.swmed.edu
Accepted: July 22, 1998.
Received: May 14, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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