The uptake of L-(methyl- 3 H) carnitine by the rat epididymis

The uptake of radioactivity by the epididymis and other tissues was measured following administration of L-[methyl-³H]carnitine to male rats. Rapid uptake occurred in both the caput and cauda epididymides. This radioactivity was shown to be present in carnitine and was located almost exclusively within the epididymal lumen.     

Neutral amino acid absorption by the rat epididymis

The technique of stopped-flow/split-drop microperfusion was used to study the absorption of the neutral amino acid alpha-aminoisobutyric acid (AIB) from different epididymal regions of the rat. Absorption of AIB from the lumen of the caput, corpus, and cauda was saturable and time-dependent. The apparent Km values for each of the regions studied were similar (approximately 6 mM), whereas the Vmax values were progressively higher from caput, corpus, and cauda, respectively. Absorption of AIB from the lumina of the caput, corpus, and cauda epididymidis was linear over 60 min. The absorption of AIB from the lumen of the caput was sodium-dependent and inhibitable by 2-methyl-alpha-aminoisobutyric acid (MeAIB), a specific inhibitor of neutral amino acid transport. Similarly, absorption of AIB from the lumen of the corpus epididymidis was sodium-dependent; however, uptake was not significantly reduced in the presence of MeAIB. Absorption of AIB from the lumen of the cauda epididymidis was neither sodium-dependent nor inhibitable by MeAIB. It is suggested that neutral amino acid absorption involves different transport carriers in different epididymal regions. These findings also support our previous observations that there exists a selective permeability barrier from lumen to blood along the epididymal duct.     

Characterization of organic cation/carnitine transporter family in human sperm

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (K(m) of 3.39+/-1.16 microM; V(max) of 0.23+/-0.14 pmol/min/mg sperm protein; and mean+/-SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (K(m) of 25.9+/-14.7 microM; V(max) of 1.49+/-1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (K(m) of 412.6+/-191 microM; V(max) of 32.7+/-20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to L-carnitine supplementation.     

Distribution of carnitine and acylcarnitine in the hamster epididymis and in epididymal spermatozoa during maturation

The highest levels of carnitine and acylcarnitine were found in the cauda epididymidis, and spermatozoa from the cauda contained greater amounts of total carnitine (free carnitine plus acylcarnitine) than those removed from the corpus or caput epididymidis. Spermatozoa from the distal cauda contained significantly greater amounts of both free and total carnitine than those removed from the proximal cauda epididymidis. The acylcarnitine:carnitine ratio was 1.7 and 0.37 in caput and cauda spermatozoa, respectively and 1.7 and 1.3 in caput and cauda fluid, respectively. It is suggested that the accumulation of carnitine is involved in sperm maturation and that acylcarnitine serves as an energy substrate for epididymal spermatozoa.     

Luminal secretion of myo-inositol by the rat epididymis perfused in vitro

A technique for perfusing the lumen of rat epididymal tubules maintained in vitro showed that [3H]inulin was largely excluded from the lumen of unravelled tubules from the cauda and tubules from the corpus if the connective tissue capsule was removed. The preparation transported [3H]inositol from the bath fluid for 3 h against a concentration gradient in both regions with activity rising (16-29% of bath fluid values) in the cauda and reaching a plateau (18%) in the corpus epididymidis. HPLC showed that radioactivity was solely associated with inositol and its movement to the lumen was reduced by raising inositol in the bath fluid from 50 microM (plasma levels) to 10 mM, but not affected by reducing the glucose concentration in the bath fluid or introducing physiological concentrations of inositol (30 mM) into the lumen. Secretion into the caudal lumen of unlabelled inositol measured by g.l.c. was maintained for 3 h at concentrations (300 microM) greater than those in the bath fluid and was not reduced when glucose or inositol were removed from the bath. In contrast, glucose was only detectable in the lumen when it was present in the bathing medium, reaching 1% of this concentration. Radioactivity appeared in the epididymal lumen reaching a plateau (19% of bath fluid values) in the corpus and cauda when [3H]glucose was added to the bath fluid, but no radiolabelled inositol was found in the lumen. We conclude that epididymal tissue is a major source of secreted inositol.     

Absorption of D- and L-carnitine by the intestine and kidney tubule in the rat

The process by which L- and D-carnitine are absorbed was investigated using the live rat and the isolated vascularly perfused intestine. A lumenal dose of 2-6 nmol in the perfused intestine resulted in less than 5% transport of either isomer to the perfusate in 30 min. The L-isomer was taken up by the intestinal tissue about twice as rapidly as the D-isomer by both the perfused intestine (52.8% and 21.6%, respectively) and the live animal (80% and 50%, respectively) in 30 min. After 1 h 90% of the L-carnitine had accumulated in the intestinal tissue and was released to the circulation over the next several hours. Accumulation of D-carnitine reached a maximum of 80% in 2 h and release to the circulations was similar to that of L-carnitine. Uptake of both L-[14C]carnitine and acetyl-L-[14C]carnitine was more rapid in the upper jejunal segment than in other portions of the small intestine. Acetylation occurred in all segments, resulting in nearly 50% conversion to this derivative in 5 min. Increasing the dose of L-carnitine reduced the percent acetylation. The uptake of both isomers was a saturable process and high concentrations of D-carnitine, acetyl-L-carnitine and trimethylaminobutyrate inhibited L-carnitine uptake. In the live animal after 5 h, the distribution of isotope from L-[14C]carnitine and D-[3H]carnitine differed primarily in the muscle where 29.5% of the L-carnitine and 5.3% of the D-carnitine was found and in the urine where 2.9% of the L-carnitine and 7.1% of the D-carnitine was found. The renal threshold for L-carnitine was 80 microM and for D-carnitine 30 microM, in the isolated perfused kidney. Approx. 40% of the L-carnitine but none of the D-carnitine excreted in the urine was acetylated. L-Carnitine and D-carnitine competed for tubular reabsorption.     

Uptake, metabolism and release of carnitine and acylcarnitines in the perfused rat liver

The uptake and release of carnitine and isovalerylcarnitine have been studied in the perfused rat liver. Labelled carnitine accumulates in rat livers perfused with 50 or 500 microM [3H]carnitine. When alpha-ketoisocaproate (5 mM) is added to the perfusate after 30 min of perfusion, the net uptake of carnitine in the liver stops, and there is even a decrease in liver radioactivity. The decrease in liver carnitine can be attributed to an enhanced formation and efflux to the perfusate of short-chain acylcarnitines. Thin-layer chromatography of liver and perfusate extracts showed that efflux rates for branched-chain acylcarnitines (isovalerylcarnitine) formed are at least 2.5-fold the efflux rate for carnitine. Acetylcarnitine is released about twice as fast as carnitine from the liver. Perfusion with 50 microM [3H]isovalerylcarnitine showed that the influx rate of isovalerylcarnitine exceeds that of carnitine 1.5-fold. Since the efflux rate is still higher, a net loss of carnitine from the liver to the perfusate will result when branched-chain acylcarnitines are formed in the perfused liver. The addition of 500 microM unlabelled carnitine to the perfusate does not influence the release of labelled carnitine or acylcarnitines from the liver, showing that uptake and release are independent processes. Isovalerylcarnitine accumulates faster than carnitine does, also in the perfused rat heart. A mechanism for the development of secondary carnitine deficiencies associated with organic acidemia is proposed.     

The distribution of carnitine and acetylcarnitine in the rabbit epididymis and the carnitine content of rabbit spermatozoa during maturation.

The highest levels of carnitine and acetylcarnitine were found in the cauda, and spermatozoa from the proximal cauda contained significantly greater amounts of carnitine than those removed from the corpus or caput epididymidis. Acetylcarnitine levels (as a % of the total carnitine pool) were greater in all regions of the rabbit epididymis than has been reported in other species. It is suggested that the accumulation of carnitine is involved in sperm maturation.     

Effects of sulphapyridine on sperm transport through the rat epididymis and contractility of the epididymal duct

This study was undertaken to investigate the effects of sulphapyridine on the transport of spermatozoa through different regions of the epididymis and on the contractility of the epididymal duct in the rat. Sperm transport was investigated by labelling testicular spermatozoa with [3H]thymidine and measuring intraluminal pressures of the epididymis by micropuncture, using a servo-nulling pressure transducer system. In control rats, the transit times of epididymal spermatozoa from the initial segment to the caput, from the caput to the proximal cauda, and from the proximal cauda to the distal cauda were 2, 6 and 3 days, respectively, giving a total transit time of 11 days. The total transit time was shortened to 8 days after treatment with sulphapyridine at a dosage of 450 mg kg-1 for 38-52 days. The rate of sperm transport was most affected in the caput epididymidis. Measurements of intraluminal pressures showed that sulphapyridine had no effect on spontaneous contractions in any regions of the epididymis. However, the frequency of contraction of the corpus and cauda epididymides in response to administration of 10 micrograms noradrenaline kg-1 in the sulphapyridine-treated rats was significantly higher (P < 0.05) than it was in the controls. Methacholine, at a dose of 20 micrograms kg-1, produced a smaller increase in basal pressure in the caput epididymidis of sulphapyridine-treated rats (P < 0.05) compared with controls. The results led to the conclusion that sulphapyridine increases the rate of sperm transport from the caput through the cauda epididymidis, in part, by changes in the responsiveness of the epididymis to the autonomic nervous system.     

Investigation by luminal perfusion of the transfer of compounds into the epididymis of the anaesthetized rat.

Controlled perfusion through the lumen of the distal cauda epididymidis in the anaesthetized rat has been explored as a means of examining physiological exchanges from blood across the epididymal epithelium. The mean length of the perfused, sperm-free, tubule was 14.5 cm (+/- 1.5 s.e.m., n = 9). No cholesterol, protein or sialic acid was detected in the perfusate at flow rates exceeding 10 microliters/min, but at rates of 0.4--1.2 microliters/min, protein appeared at concentrations of 0.21--0.55 mg/ml (i.e. secretion rates of 0.21--0.83 micrograms/min; 3 rats). Glucose was detected at all perfusion rates (3--27 microliters/min) at concentrations of 0.06--0.58 mM (0.8--6.8% blood levels). During intravenous infusions of 3H2O, radioactivity in the perfusate rapidly attained 87% blood plasma concentrations; no radioactivity was detected when carboxy-E114C]dextran or methoxy-[3H]inulin were infused. Radioactivity appeared in the epididymal perfusate to 1--7% of blood levels during intravenous infusions of D-E1U-1RC]glucose or 3-O-methyl[1-3H]glucose. This evidence suggests that the preparation is physiological and could be used to explore the dynamics of exchanges between blood and epididymis.     

Carnitine transport and exogenous palmitate oxidation in chronically volume-overloaded rat hearts

L-Carnitine transport and free fatty acid oxidation have been studied in hearts of rats with 3-month-old aorto-caval fistula. For carnitine transport experiments, the hearts were perfused via the ascending aorta with a bicarbonate buffer containing 11 mM glucose and variable concentrations L-[14C]carnitine (10-200 microM). In some experiments, the active component of carnitine transport was suppressed by the adjunction of 0.05 mM mersalyl acid. The subtraction of passive from total transport allowed reconstruction of the saturation curves of the carrier-mediated transport of L-carnitine. Our data suggest that at a physiological carnitine concentration (50 microM), the rate of [14C]carnitine accumulation was significantly depressed in mechanically overloaded hearts. In addition, according to Lineweaver-Burk analysis, the affinity of the membrane carrier for L-carnitine was considerably diminished (Km carnitine 125 instead of 83 microM, Vmax unchanged). The above alterations of L-carnitine transport did not result from a decrease of the transmembrane gradient of sodium, since the intracellular Na+ content of the hypertrophied hearts was quite similar to that of control hearts. The ability of atrially perfused, working hearts to oxidize the exogenous free fatty acids was assessed from 14CO2 production obtained in the presence of [U-14C]palmitate or [1-14C]octanoate. The total 14CO2 production, expressed per min per g dry weight, was significantly diminished in hearts from rats with the aorto-caval fistula if 1.2 mM palmitate was used. On the other hand, in the presence of 2.4 mM octanoate, a substrate which circumvents the carnitine-acylcarnitine translocase, no such reduction of the 14CO2 production could be detected. Our results suggest that the decrease of L-carnitine transport, resulting in a significant depression of tissue carnitine, may impair long-chain fatty acid activation and/or translocation into mitochondria. In contrast, the oxidation of short-chain fatty acids, the activation of which takes place directly in mitochondrial matrix, is not limited in volume-overloaded hearts.     

Ultrastructure of the perfused rat epididymis: Effect of luminal sodium ion concentration

Summary The appearance of the rat epididymal epithelium changed when it was perfused in vivo through the lumen with unphysiologically high sodium ion concentrations; dilatation of intercellular spaces (ICS) at threshold concentrations of 30mM-Na⁺ in the cauda and about 55mM-Na⁺ in the corpus was associated with absorption of water from the lumen. Despite the distended ICS, junctional complexes appeared intact, and their integrity was confirmed by the exclusion of luminal horseradish peroxidase (HRP) from the ICS, and by demonstrating that circulating [³H]inulin did not enter the lumen. Smooth ER and lipid droplets in the principal cells of the corpus epididymidis were well maintained, and the preservation of granular ER in principal cells of the cauda epididymidis lent morphological support to the continued secretion of protein in this segment. However, occasional distension or involution of inner Golgi cisternae was evident in principal cells after 3–6 h perfusion. In contrast to multivesicular bodies of principal cells, the apical and basal vacuoles characteristic of clear cells changed in size with different perfusing solutions. When low Na⁺ concentrations were perfused large translucent vacuoles were frequently found in the apical cytoplasm of clear cells in the corpus and cauda epididymidis, and filled vacuoles became larger and showed a decrease in content density in the cauda epididymidis. These large vacuoles were absent from tissue perfused with high Na⁺ concentrations. Normal pinocytotic activity of both cell types was demonstrated by perfusing HRP which was taken up by the normal route in principal cells, with some transfer to the Golgi cisternae. By far the most HRP was accumulated in clear cell vacuoles irrespective of the composition of the perfusing solution.     

Metabolism and excretion of carnitine and acylcarnitines in the perfused rat kidney

Rat kidneys were perfused for 30 min with a Krebs-Henseleit bicarbonate buffer with 5 mM glucose. Albumin proved superior to pluronic polyols as oncotic agent with regard to carnitine reabsorption in the perfused kidney. The reabsorption of 30 μM (−)-[methyl-³H]carnitine was approx. 96% during the first 10 min. At 750 μM the reabsorption decreased to 40%. The tubular reabsorptive maximum (T_max) was approx. 170 nmol/min per kidney. The fractional reabsorption and clearance of (+)-carnitine, γ-butyrobetaine, and carnitine esters did not deviate significantly from that of (−)-carnitine. (+)-Carnitine was not metabolized by the perfused kidney. In perfusions with (−)-carnitine or (−)-carnitine plus 10 mM α-ketoisocaproate or α-ketoisovalerate increased amounts of acetylcarnitine, isovalerylcarnitine and isobutyrylcarnitine were found. Propionate (5 mM) inhibited acetylcarnitine formation. Isovalerylcarnitine, isobutyrylcarnitine and propionylcarnitine were actively degraded to free (−)-carnitine. In urine, we found a disproportionally high excretion of carnitine or carnitine esters formed in the kidney, compared to the same derivatives when ultrafiltrated. Leakage of metabolites formed in the kidney into preurine may explain this phenomenon.     

Intraluminal androgen binding protein alters 3H-androgen uptake by rat epididymal tubules in vitro

Previous experiments have shown that androgen binding protein (ABP) and androgens exist in high concentrations in the tissue and the lumen of the rat caput epididymis. The present experiments were performed to determine whether or not intraluminal APB affects tubule net uptake of androgens. Caput epididymal tubules were dissected into 2-cm segments, subjected to microperfusion into the tubule lumen, and incubated for 2.5 h in 35 degrees C minimum essential medium (MEM) containing 2.0 ng tritiated testosterone (3H-T) per ml. 14C-polytheylene glycol [PEG] was included as a contamination marker. In the first series of experiments, caput tubules were perfused with a control, artificial perfusion (MKB) containing no ABP or fresh rat rete testis fluid (RTF), which is known to contain ABP. Tubules incubated while containing RTF took up 138% of the tritiated androgens taken up by control tubules. In the second series of experiments, tubules were perfused with fresh caput epididymal lumen content, MKB alone, MKB containing either 5.0 ng purified rat ABP/microliters or 50 ng ABP/microliters. Tubules incubated while containing perfused MKB took up only 47% of the tritiated androgens taken up by tubules containing perfused native lumen content. Increasing intraluminal ABP concentrations in the MKB medium increased 3H-androgen uptake in a stepwise fashion. Intraluminal ABP at a concentration of 50 ng/microliters was associated with a 71% return of 3H-androgen uptake towards that amount of 3H-androgen taken up by tubules perfused with native lumen content. Intraluminal ABP enhances net androgen uptake by caput epididymal tubules from their surrounding medium in vitro.     

Enhancement of sperm transport through the rat epididymis after castration

Transport of spermatozoa through different regions of the epididymis has been followed by labelling testicular spermatozoa with [3H]thymidine in intact rats and in rats in which the efferent ducts were ligated or the testes were removed. In intact rats, the transit times of epididymal spermatozoa from the initial segment to the caput, from the caput to the corpus, and from the corpus to the cauda were 2, 4 and 2 days, respectively, giving a total transit time of 8 days. After bilateral castration, labelled spermatozoa were transferred from the initial segment into the proximal cauda by 2 days and appeared in the ductus deferens by 4 days. This effect was prevented by a daily subcutaneous injection of testosterone propionate (0.2 mg/kg). Bilateral efferent duct ligation had only a slight effect on the passage of epididymal spermatozoa. The results indicate that epididymal sperm transport is enhanced after androgen withdrawal.     

Role of epithelial clear cells of the rat epididymis in the disposal of the contents of cytoplasmic droplets detached from spermatozoa

Upon release from the seminiferous epithelium, spermatoza show a small droplet of cytoplasm attached to the neck region. During transit of spermatozoa in the caput epididymidis, this cytoplasmic droplet migrates along the middle piece of the flagellum. In the corpus epididymidis, the droplet shows a lateral displacement, while in the cauda epididymidis it detaches from the spermatozoon. In the electron microscope, cytoplasmic droplets attached to spermatozoa were seen to contain numerous, short, straight or C-shaped, flattened membranous elements referred to as lamellae, small vesicles, and small particles (35-nm diameter) with a diffuse wall showing no apparent unit membrane. The lamellae were stacked closely on one another or arranged in a loose array. Structurally as well as cytochemically, with different cytochemical markers, the lamellae and vesicular elements failed to show any evidence of being components of the Golgi apparatus or elements of the endoplasmic reticulum. The lamellae, vesicular elements, and 35-nm particles were also seen free in the lumen of the corpus epididymidis but were especially prominent in the cauda epididymidis at a time when droplets were being released from spermatozoa. The lumen of the epididymis, as spermatozoa passed from the caput to the cauda epididymidis, was also noted to acquire progressively a flocculent background material. The epididymal epithelium is composed predominantly of principal and clear cells. The endocytic activity of clear cells was examined in rats at different time intervals after a single injection of cationic ferritin into the lumen of the cauda epididymidis. At 2 min the tracer was bound to the microvilli of these cells and was also observed within large coated and uncoated pits, subsurface coated vesicles, and numerous subsurface small uncoated vesicular membranous elements (150-200-nm diameter). At 5 min, in addition to the above structures, the tracer was present in endosomes, while at 15 and 30 min, pale and dense multivesicular bodies appeared labeled, respectively. At 1 and 2 hr, but more so at 6 hr large dense membrane-bound bodies identified cytochemically as secondary lysosomes became labeled. All of the above endocytic structures were also seen to contain the 35-nm particles, flattened or vesicular membranous profiles, and a fine flocculent background material reminiscent of those seen free in the lumen or found in cytoplasmic droplets attached to spermatozoa. (ABSTRACT TRUNCATED AT 400 WORDS)     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose

Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calcium (1 mM) was added at T = 15 min, and heparin (HEP; 10 micrograms/ml) or heparin plus glucose (hGLUC; 5 mM in 10 micrograms/ml heparin) was added at T = 30 min. Spermatozoal Ca2+ content and regulation differed among males (P = 0.0066). Relative initial [Ca2+]i differed significantly among all stages of maturity (0.84 +/- 0.104, 0.76 +/- 0.023, 1.20 +/- 0.036 LSM of relative Ca2+ units for caput, cauda and ejaculate spermatozoa respectively; P = 0.001). Rate of Ca2+ uptake was similar for ejaculate and cauda spermatozoa (0.021 +/- 0.005 and 0.026 +/- 0.002 relative Ca2+ units/sec) but slower for caput spermatozoa (0.012 +/- 0.001; P = 0.0006). There was no immediate effect of HEP or hGLUC in any stage (P > 0.05), and caput spermatozoa did not differ from cauda spermatozoa for any treatment or time period. A significant increase in [Ca2+]i was seen in ejaculate spermatozoa treated with HEP from 2 h on (P < 0.05). This study demonstrates that both the absolute Ca2+ concentration and the rate of Ca2+ internalization in equine spermatozoa is dependent on the stage of maturation. Ejaculate spermatozoa respond to heparin through increased [Ca2+]i, which may play a role in the fertilizing ability of ejaculate spermatozoa.