A factor in human seminal plasma which affects carnitine accumulation in bovine epididymal sperm*t

Transcription

1 FERTILITY AND STERILITY Copyright <> 1988 The American Fertility Society Printed in U.S.A. A factor in human seminal plasma which affects carnitine accumulation in bovine epididymal sperm*t A. Lee Carter, Ph.D.:j: Soo H. Cho, M.D.II1f Elisabeth R. Bishop, B.S.:j: Jeffrey Boldt, Ph.D.11 Medical College of Georgia, Augusta, Georgia This study was initiated to determine whether factors are present in human sperm-free seminal plasma (HSP) that regulate the uptake and release of carnitine from sperm. Bovine caput epididymal sperm cells accumulated more carnitine than caudal sperm cells. A significant reduction in carnitine uptake by caput sperm was observed in the presence of HSP from normal subjects, but not from three subjects with reduced motility. A factor has been isolated from HSP that inhibits carnitine uptake by caput sperm and has the following properties: it is nondialyzable, stable to freeze-thawing, soluble in 60% ammonium sulfate, and has an approximate molecular weight of 158 kd. These data are consistent with the existence of a relatively high molecular weight protein in HSP responsible for the preservation of carnitine concentrations in sperm. Fertil Steril 49:893, 1988 Carnitine is a component of both seminal plasma and sperm cells. In seminal plasma, the carnitine concentration is approximately 0.32 mm, 1 while in sperm the concentration can be more than 20 times that of seminal plasma. 2 Its main function appears to be in the storage of "acetate moieties" derived intracellularly from acetylcoenzyme A.3 Under usual circumstances, more than 90% of the carnitine in sperm is present as acetylcarnitine. 4 This acetylcarnitine is converted to free carnitine and the acetate moiety is used as an energy source Received September 1, 1987; revised and accepted January 6, * This is publication 1056 from the Department of Cell and Molecular Biology, Medical College of Georgia. t Supported in part by the National Institutes of Health grant AM and by the Muscular Dystrophy Association. :: Department of Cell and Molecular Biology. Reprint requests: A. Lee Carter, Ph.D., Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, Georgia II Department of Obstetrics and Gynecology, Section of Reproductive Endocrinology. 11 Present address: Department of Obstetrics and Gynecology, School of Medicine, Hanyang University, Seoul, Korea. under conditions where motility is required with oxygen present but limited sources of external energy.5 In dairy bulls, a direct correlation has been shown between sperm carnitine levels and fertility.3 Oligospermic humans have low carnitine levels.s The acetylcarnitine concentration in sperm is positively correlated with human sperm motility, but not with sperm density.7 Using micropuncture studies, it has been shown that carnitine levels in epididymal fluid and sperm cells dramatically increase as sperm are transported through the epididymis.s Caudal sperm have much higher levels of carnitine than caput sperm. 9 Carnitine is readily accumulated by bovine caput sperm/a but it is accumulated at a slower rate by caudal epididymal sperm. Externally added carnitine is not accumulated by ejaculated bovine ll or human sperm. 12 The concentration of carnitine in ejaculates of dairy bulls is positively correlated with plasma testosterone levels3 and, in rats, carnitine uptake by the epididymis is shown to be androgen-dependent.13,14 Johansen and Bohmer15 suggest that the presence of a factor in the acces- Carter et al. Seminal plasma effect on carnitine uptake 893

2 sory male sex organs increases the carnitine binding sites in bovine caudal sperm. It is the aim ofthis study to determine whether there are factors in human seminal plasma that may affect carnitine accumulation, thereby decreasing the motility and the fertilizing capacity of sperm. Materials MATERIALS AND METHODS I4C(methyl)-L-carnitine was purchased from Amersham (Chicago, IL). All other chemicals were purchased either from Fisher (Atlanta, GA) or Sigma Chemical Company (St. Louis, MO), and were either reagent grade or the best grade available. Semen Analysis and Preparation of Human Seminal Plasma Samples were routinely received from the reproductive endocrinology clinic for semen analysis. The following parameters were recorded using the World Health Organization criteria: semen volume, sperm count, motility, progression, and morphologic features. After determining these seminal parameters, sperm were pelleted by centrifugation at 600 X g for 10 minutes in a clinical centrifuge at room temperature, and the sperm-free seminal fluid (HSP) removed and frozen at -20 C until processed and assayed. When three to six samples had been collected, they were dialyzed in individual dialysis bags against 5 liters of phosphate-buffered saline (PBS) overnight with two buffer changes. This procedure removed carnitine and other small molecules that could have interfered with the carnitine uptake experiments. After dialysis, these HSP samples were either frozen at -20 C or assayed before freezing to determine their effect on carnitine uptake by bovine epididymal sperm. Preparation of Epididymal Sperm for Uptake Studies Bovine testicles were obtained from a local slaughterhouse within 10 minutes after death, kept on ice, and delivered to the laboratory within 1 hour. Upon arrival in the laboratory, the epididymis was removed and the caudal and caput portions separated and placed in separate beakers containing enough PBS to cover the epididymis. After several incisions were made on the ventral surface of the specimen, it was incubated for Carter et al. Seminal plasma effect on carnitine uptake minutes at 37 C and sperm was released into the PBS medium. Sperm were collected by centrifugation in a clinical centrifuge at 400 X g for 5 minutes. The sperm pellet was resuspended in PBS and the centrifugation repeated. After a second washing, the motility was determined, a sperm count made, and these cells used for the measurement of carnitine accumulation. Carnitine Assays These assays were performed as previously described. 16 Briefly, to a 1-ml sperm solution of known cell count or to seminal plasma diluted to 1 ml was added 0.1 ml of concentrated perchloric acid (11.8 N). After vortex (Fisher, Atlanta, GA) mixing and centrifugation (10,000 X g for 5 minutes), the supernatant was transferred to a new centrifuge tube. The pellet was extracted with 0.5 ml of 1 N perchloric acid. This extract was combined with the supernatant. To this combined solution was added 0.5 ml of 8 N potassium hydroxide. After 1 hour, the solution was centrifuged (10,000 X g for 5 minutes) to pellet insoluble salts. The supernatant was transferred to a test tube and incubated for 10 minutes in a boiling water bath and then cooled in ice. The ph was adjusted to between 6 and 8 with hydrochloric acid, and these samples were stored until the time of assay. The carnitine assay was carried out in 1.5-ml microfuge tubes (Fisher, Atlanta, GA) with water and sample added to equal 0.1 ml. A standard curve of L-carnitine (Sigma Tau, Pomezia, Italy) from 20 to 60 pmol was constructed and run with each assay. To each of the microfuge tubes in a volume of 0.1 ml was added either standard, unknown, or water (for a blank), and 0.1 ml of a solution containing the following: 0.1 M TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2 mm N-ethyl-maleimide, I4C-acetyl-CoA, and five units of carnitine acetyl transferase. These tubes were mixed and incubated exactly 60 minutes at room temperature. The reaction was terminated by the addition of a 0.6 ml slurry of Dowex 1 X to 400 mesh equivalent to 80 mg of dry resin. The samples then were mixed, placed in ice for 30 minutes, and mixed two additional times during this period. Next, the microfuge tubes were centrifuged for 1 minute in a microfuge (Brinkman Inst., Westbury, NY) and 0.2 ml of the supernatant counted in a Beckman LS 230 scintillation counter (Irvine, CA). Disintegrations per minute (DPMs) were determined using the external standard ratio method. Fertility and Sterility

3 From the carnitine standards, a linear regression was calculated for DPMs versus carnitine added, and the unknown samples calculated from this analysis. Carnitine Uptake Studies The final sperm count was adjusted to 2 X 10 8 cells/ml. One half milliliter of the sperm suspension was added to a 0.5 ml solution containing PBS, 0.4 JLCi of 14C-carnitine, and other additions, as specified. Samples were incubated for 45 minutes at 37 C unless otherwise specified. At the end of the incubation, the samples were immediately centrifuged for 1 minute in an Eppendorf microfuge (Brinkman Inst., Westbury, NY) and the supernatant removed. The pellet was resuspended in 1 ml of PBS and centrifuged as described. This washing procedure was repeated two additional times. The final sperm pellet was suspended in 0.2 ml of PBS, transferred to scintillation vials containing 5 ml of scintillation fluid (Scintiverse E, Fisher, Atlanta, GA), and the samples counted in a Beckman LS 230 scintillation counter. DPMs were determined by the external standard ratio method. Ammonium Sulfate Fractionation The dialyzed seminal plasma was pooled and placed in a beaker with a magnetic stirrer in an ice bath. Solid ammonium sulfate (176 mg/ml) was added over a 30-minute period to obtain a saturation of 30%. After the final addition of the ammonium sulfate, stirring was continued for 30 minutes and the sample centrifuged for 10 minutes at 10,000 X g. The supernatant was decanted into a graduated cylinder and the volume recorded. The pellet was resuspended in a volume of 5 ml, labeled as the 0% to 30% fraction, and dialyzed with the subsequent fractions for 24 hours against 5 I of PBS with two buffer changes. The supernatant was made 45% saturated with the addition of 61 mg/ml of ammonium sulfate over a 30-minute period. Stirring was continued for 30 minutes after the addition of the ammonium sulfate was completed, and the 30% to 45% pellet was separated from the supernatant and dialyzed as described. The supernatant then was made 60% saturated by the addition of 97 mg/ml of ammonium sulfate over 30 minutes and stirring was continued for an additional 30 minutes. The 45% to 60% pellet then was separated from the supernatant and dialyzed as described. The volume of the supernatant was recorded, labeled as the ammonium sulfate soluble fraction, and dialyzed as described. After dialysis, the fractions were stored overnight at 5 C and then assayed for their effect on carnitine uptake, and the amount of protein present was determined.17 After assaying, the remaining samples were stored at -20 C. Determination of Approximate Molecular Weight The ammonium sulfate soluble fraction was concentrated by an Amicon concentrator with a PM -30 membrane to a volume of approximately 5 ml and dialyzed overnight against 5 liters of PBS. A portion (5 JLI, 120 JLg of protein) was applied to a TSK-GEL-3000SW molecular sieve column (Phenomenex, Rancho Palos Verdes, CA) highperformance liquid chromatography equilibrated with LCB buffer (50 mm sodium phosphate and 0.2 M sodium chloride, ph 7.4), and the column was eluted with LCB at a flow rate of 1 mllmin by a Gilson gradient high-performance liquid chromatography system (Gilson Medical Electronics, Middleton, WI). Protein concentrations were determined by continuous monitoring at 280 nm. Fractions found in protein peaks were combined, dialyzed overnight against PBS, and assayed for their effects on carnitine uptake. Analysis of Data Results are presented as mean ± the standard error of the mean with significance determined by the Student's t-test. With nonparametric data (data expressed as percentage of control), nonparametrical analysis was performed using the Whitney-Mann nonparametric test of significance. RESULTS Typical motility in bovine caudal and caput sperm samples prepared as described were approximately 40 and 3%, respectively. These preparations were essentially free of cellular debris and other cell types. Usually more than 10 9 cells were obtained from two epididymides for each of the sperm preparations. The accumulation of carnitine in both the caudal and caput specimens increased for the first 45 minutes of incubations, as shown in Figure 1. For the remainder of the experiments, 45 minutes was used for the incubation time, unless otherwise specified. While caudal sperm accumulated small-but-significant amounts of carnitine, the accumulation of carnitine was much greater by caput sperm. Using the procedure described in Ma- Carter et al. Seminal plasma effect on camitine uptake 895

4 ::;: " / L_ J! l, /"... / """,,1,/ CAPUT TIME (MINUTES) Figure 1 Accumulation of carnitine by bovine epididymal sperm. Each point is the mean ± the standard error of the mean for four separate determinations performed in triplicate. terials and Methods, the amount of radioactivity in the third wash was less than 10% ofthe radioactivity found in the sperm cells (data not shown). The zero time point samples had significant amounts of radioactivity (about 500 DPM), which could either represent rapid uptake of carnitine during centrifugation or binding of carnitine to the sperm plasma membrane. To determine the effects of HSP on carnitine accumulation, 25 consecutive seminal plasma samples were obtained after semen analysis for possible male infertility. The seminal plasma was separated from sperm by centrifugation and stored at -20 C until assayed. No sperm was present under microscopic examination. Sperm cells from two to six epididymides, as available, were combined to obtain enough sperm cells for carnitine uptake studies. Each seminal plasma sample was run in triplicate with either 0.2 ml of seminal plasma or 0.2 ml of PBS added to each sample for assay. The average of these means for 25 consecutive patients is presented in Table 1. While a large amount of variation existed among the patients, there was a significant decrease in the accumulation of carnitine in the presence of HSP by caput sperm, and a small-but-significant increase by caudal sperm (Table 1). For comparison of individual seminal plasma samples, the uptake of carnitine was converted to percent of control. A control assay was run with each seminal plasma sample. This corrects for the large day-to-day variation in carnitine uptake by epididymal sperm obtained from different bulls. The comparison of the percentage of carnitine uptake as compared with controls to the various seminal parameters obtained upon collection is shown in Table 2. Sperm progression and morphologic features (not shown) were not statistically significantly related to the presence of the carnitine "inhibitor." The seminal parameters listed in Table 2 were divided into normal and abnormal groups, respectively, as follows: semen volume, greater and less than 1.0 ml; sperm concentration, greater and less than 20 X 10 6 cells/ml; and motility, greater and less than 40% motile cells. In the comparison ofthe inhibition of uptake of carnitine by seminal fluid to semen parameters, the only parameter that gave a significant difference by the Whitney-Mann U-test was that of motility (Table 3). From Table 2, note that there were 21 normal and 4 patients with low motility. No other differences were found between the effects of seminal plasma on carnitine uptake by caudal sperm and seminal parameters listed in Table 2. The pooled seminal plasma was thawed and subjected to ammonium sulfate fractionation as described in Materials and Methods. The dialyzed fractions were assayed for activity and protein. For the purpose of purification, one unit of activity was arbitrarily defined as the amount of the "factor" required to inhibit the uptake of carnitine of 10 8 bovine caput epididymal cells by 50%. In terms of total activity, there were 126 units of activity in the pooled seminal plasma used for the ammonium sulfate fractionation procedure. No inhibition of carnitine uptake was obtained by fractions that precipitated at ::;45% ammonium sulfate. The 45% to 60% fraction had 36.5 units of activity, while the ammonium sulfate supernatant fraction had 87 units. The initial specific activity was U /mg protein, while the specific activities in the 45% fraction and the ammonium sulfate supernatant were 2.4 and 2.6, respectively. This represents a Table 1 The Effect of Seminal Plasma on the Uptake of 14C-Carnitine in Cauda and Caput Epididymal Sperm Cauda Caput seminal seminal PBsa plasma PBS plasma Uptake b ' ' (351) (460) (584) (536) a Phosphate-buffered saline. b Uptake measured in DPMs with the standard error of the mean in parentheses., P < 0.05 versus PBS by the paired Student's t-test for n = 25 pairs. 896 Carter et al. Seminal plasma effect on carnitine uptake Fertility and Sterility

5 Table 2 Comparison of Carnitine Uptake According to the Semen Parameters in Individuals No. Concentrationa Motility" Xlo'lml %Iml a Mean for three determinations. Volumea Caputa,b Caudaa,b ml b Each value is the percentage of the means compared with the control mean (ie., incubated in PBS alone). protein purification of approximately 8-fold. Since most of the activity was recovered in the ammonium sulfate supernatant fraction, this material was used for the further characterization of the protein, as described subsequently. Analysis of the 60% soluble fraction by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis revealed more than 15 bands (not shown), which indicates that additional steps are required for the complete isolation of this inhibitor. Up to this point, the Table 3 Comparison of Carnitine Uptake in Normal and Asthenospermic Group Normal Cauda Percent b change 118 ± 7' 158 ± 45 d a Motility ~40%. Normal 68 ± 7' Caput b Percent of control ± the standard error of the mean. 'n = 21. d n = 4. e p < 0.05 by the Mann-Whitney U-test. Asthenospermica Asthenospermica 98 ± 10 d " samples have been stored for up to 1 month at -20 C, dialyzed several times, and frozen and thawed several times with no apparent loss in activity. The data in Figure 2A indicates that increasing amounts of the 60% ammonium sulfatesupernatant give a linear decrease in carnitine uptake over a wide range. This allowed us to define a unit of material as the amount of the "inhibitor" required to give 50% inhibition of carnitine uptake by caput sperm. An apparent molecular weight of 158 kd was obtained by comparison of the position of elution of the carnitine "factor" to that of known standards on a TSK-3000SW molecular sieve HPLC column (Fig. 2B). DISCUSSION The accumulation of carnitine was greater in caput sperm cells than caudal cells, which is similar to a previous report.lo The accumulation of carnitine in these experiments is believed to be by internalization into cells and not simply by binding. Our washing procedure is similar to that of Casillas,lO and we observed that three washings with PBS Carter et al. Seminal plasma effect on carnitine uptake 897

6 500 A B A 620)( i "K " n", 1,f 1 ""i 1 1", j.1 I,! /; "/ i """,I, ""f ~. 100 zoo ONIU.. SUlfATE SUPERNATANT (ul) "t-o-',~,-':-~w'--',c:-, 7.,,'--',', -':,,'--',C::- o -::':,,--:,c,",,--;,c::-,-':,,--:,,;:-,""--:,c--;,, ". FRACTION NUMBER Figure 2 Characterization of the carnitine inhibitory factor. (A) Inhibition of carnitine uptake by increasing amounts of partially purified human seminal plasma. Each point represents the mean ± the standard error ofthe mean. (B) Determination ofthe apparent molecular weight of the human seminal plasma "factor." The dotted line is the profile of the standard molecular weight markers as follows: vitamin B12, 1.35 kd; horse myoglobin, 17 kd; chicken ovalbumin, 44 kd; bovine gamma globulin, 158 kd; bovine thyroglobulin, 670 kd. The arrow represents the point of maximum inhibition of carnitine uptake by the "factor." readily removed carnitine. In experiments designed to measure the binding of carnitine to bovine epididymal cells, it has been shown that only very small amounts of binding occurs. 15 Under the conditions of our experiments, this binding could account for only about 10% of the amount of labeled carnitine that is observed in the sperm pellets of our experiments. Also, the accumulation of carnitine continues to increase over 45 minutes, which seems to be a long time if the increase of radioactivity is simply due to binding. In a similar pattern to the relationship of acetylcarnitine to motility,7 there is an inverse relationship between the amount of inhibition and motility of the specimen. Out of 25 specimens received for infertility, the inhibitor appeared to be missing in 7 specimens, including those with low motility. Nonparametric statistics were required for this analysis since the data are presented in Table 2 as percent of controls. The data were presented in this fashion because of a large amount of day-to-day variability in the amount of carnitine accumulation in epididymal sperm. This variation is probably due to the differences in the age and physical condition of the bulls being processed at the meat packing plant. With the partial purification of the inhibitor of carnitine uptake, we have shown that this is a factor of relatively high molecular weight (158 kd), which is a soluble protein found in human seminal plasma. The properties of this factor should allow it to be obtained in a relatively pure form for future studies. The role of carnitine in sperm cells seems to be critical. Caput sperm cells have low carnitine levels, but these levels increase rapidly as sperm mature in the epididymis. Under anaerobic conditions in the epididymis, the carnitine becomes acetylated to the point where most species contain more than 50 mm acetylcarnitine in their mature epididymal sperm. This acetylcarnitine is required for motility.5-7 Ejaculated sperm must preserve their acetylcarnitine for the motility required during the fertilization process.1s The protein described in this report is an inhibitor of the uptake of carnitine by caput epididymal sperm cells, and probably functions in ejaculates to prevent the entry, exchange, or exit of carnitine from sperm cells. This is important in the prevention of intracellular loss of acetylcarnitine. Since carnitine is required for motility, the absence ofthis factor may be one of the causes of low motility. Further studies will result in the isolation and characterization of this protein. Once these experiments are completed, the site of biosynthesis and excretion of this protein will be identified. These studies will require a different species so that tissues will be readily available from different parts of the male reproductive tract. Other studies will involve the mode of action of the inhibitor. The inhibitor may either bind carnitine very tightly or interact with the sperm plasma membrane in such a manner as to prevent the uptake of carnitine. Of particular interest will be factors that regulate the levels of this protein. Acknowledgments. We appreciated the technical assistance of Mrs. Anita Howe. We express thanks to Sigma Tau Pharmaceutical Company, Pomezia, Italy for L-carnitine and to Shapiro Packing Company, Augusta for bovine sperm. 898 Carter et al. Seminal plasma effect on carnitine uptake Fertility and Sterility

7 REFERENCES 1. Frenkel G, Peterson RN, Davis JE, Freund M: Glycerylphosphory1choline and carnitine in normal human semen and in postvasectomy semen: differences in concentrations. Fertil Steril 25:84, Hutson SM, Van Dop C, Lardy HA: Pyruvate metabolism in bovine epididymal spermatozoa. J Bioi Chem 252:1303, Carter AL, Hutson SM, Stratman FW, Henning RV Jr: The relationship of carnitine, acy1carnitine, and testosterone in the fertilizing capacity of ejaculated sperm in dairy bulls. Bioi Reprod 23:820, Suter DA, Holland MK: The concentration of free L-carnitine and L-O-acetylcarnitine in spermatozoa and seminal plasma of normal, fresh, and frozen human semen. Fertil Steril 31:541, Milkowski AL, Babcock DF, Lardy HA: Activation of bovine epididymal sperm respiration by caffeine: its transient nature and relationship to the utilization of acetylcarnitine. Arch Biochem Biophys 176:250, Kohengkul S, Tanphaichitr V, Muangmun V, Tanphaichitr N: Levels of L-carnitine and L-O-acetylcarnitine in normal and infertile human semen: a lower level of L-O-acetylcarnitine in infertile semen. Fertil Steril 28:1333, Johansen L, Bohmer T: Motility related to the presence of carnitine/acety1carnitine in human spermatozoa. Int J Androl 2:202, Hinton BT, Setchell BP: Concentration and uptake of carnitine in the rat epididymis: a micropuncture study. In Carnitine Biosynthesis, Metabolism, and Functions, Edited by RA Frenkel, JD McGarry. New York, Academic Press,1980, p Brooks DE, Hamilton DW, Mallek AK: Carnitine and glyceryl-phosphorylcholine in the reproductive tract ofthe male rat. J Reprod Fertil 36:141, Casillas ER: Accumulation of carnitine by bovine spermatozoa during maturation in the epididymis. J Bioi Chem 248:8227, Carter AL, Stratman FW: Unpublished data 12. Bohmer T, Johansen L: Inhibition of sperm maturation through intervention of the carnitine system. Int J Androl 2(Suppl):565, Marquis NR, Fritz IB: Effects of testosterone on the distribution of carnitine, acetylcarnitine, and carnitine acetyltransferase in tissues ofthe reproductive system ofthe male rat. J Bioi Chem 240:2197, Bohmer T, Hansson V: Androgen dependent accumulation of carnitine by rat epididymis after injection of 3[H]_ butyrobetaine in vivo. Mol Cell Endocrinol 3:103, Johansen L, Bohmer T: Carnitine-binding related suppressed oxygen uptake by spermatozoa. Arch Androl 1:321, Carter AL, Bishop ER, Frenkel RA, Braver H, Chuang AH: Methods for assaying carnitine and carnitine biosynthetic enzymes. In Clinical Aspects of Human Carnitine Deficiency, Edited by PR Borum. New York, Pergamon Press, 1986, p Lowry OH, Rosebrough NJ, Farr AI, Randall RJ: Protein measurement with the Folin phenol reagent. J Bioi Chem 193:265, Makler A: Evaluation and treatment ofthe infertile male. In Reproductive Failure, Edited by AH DeCherney. New York, Churchill-Livingstone, 1986, p 51 Carter et al. Seminal plasma effect on carnitine uptake 899