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HPLC DETERMINATION OF AN OXYTOCIN-LIKE PEPTIDE PRODUCED BY ISOLATED GUINEA PIG LEYDIG CELLS: STIMULATION BY ASCORBATE
M. A. KUKUCKA and H. P. MISRA Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/mL LH for 24 h in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum, and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high-performance liquid chromatography with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells 1.23, 77.4 13.8, 74.2 f stimulated with 0, 25, and 50 pM ascorbate produced and secreted 40.1 26.3 pg of an oxytocin-like peptide, respectively, per 1 x lo6 cells. These results indicate that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo and that low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro
+
+
Key Words: Oxytocin; Leydig cells; Ascorbate; HPLC; Testis; Sodium ascorbate; Peptide.
INTRODUCTION Classically, the neurohypophysial peptide hormone oxytocin has been recognized to be associated with parturition and milk ejection. In the early 1980s, the identification of a nonneural source of oxytocin secreted in significant amounts from the ovarian corpus luteum of ruminants [21, 221 rekindled a new interest in this octapeptide hormone. Shortly following the demonstration of an ovarian source of oxytocin, the presence of an oxytocin-like peptide located in the testes was reported [ 141. Using an immunocytochemical stain, approximately 80% of the intertubular (interstitial) cells, in contrast to the intratubular (seminiferous tubule) cells, immunoreacted in a manner suggesting that the interstitial Leydig cells were the source of an oxytocin-like peptide [5]. There are several indirect lines of scientific evidence that support the biosynthesis of a testicular oxytocin-like peptide de novo. Rehbein et al. [19] reported that small amounts of an oxytocin mRNA were present in rat testes. Nicholson et al. [I31 demonstrated the disappear-
From the Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. This study was presented in part at the Third Research Day Proceedings held at the Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, May 23-24, 1991. The published proceedings (No. 23919419) were catalogued with the Online Computer Library Center (OCLC) database on June 11, 1991. Address correspondence to Hara P. Misra, Professor, Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061. ARCHIVES OF ANDROLOGY 29: 185-190 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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ance of testicular oxytocin in rats following treatment with ethan- 1,2-dimethanesulfonate (EDS), which selectively depletes the Leydig cell population. Furthermore, immunoreactive oxytocin was reported to be concentrated in Leydig cell preparations isolated from dissociated rat testes [23]. Although the scientific evidence indicates that oxytocin is located in the Leydig cells, it is not certain whether a Leydig cell oxytocin-like peptide is biosynthesized de novo or is taken up and stored by the cell from some other intra- or extratesticular source [18]. In the present study, we have determined that highly purified populations of adult guinea pig Leydig cells incubated with 100 ng/mL LH produce an oxytocin-like peptide and that sodium ascorbate augments the de novo production of this peptide in vitro.
MATERIALS AND METHODS Animals. Sexually mature Dunkin-Hartley male guinea pigs weighing between 800 and 1200 g were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA). The guinea pigs were housed three to a cage in a temperature- (21 "C) and humidity- (55 %) controlled vivarium with a photoperiod of 12 h light: 12 h dark according to the Guide fur the Care and Use ofLaboratoly Animals [15]. Animals were provided a balanced NRC [16] commercially pelleted guinea pig formula diet (Agway, Inc., Syracuse, NY) and water ad libitum. Lpydig Cell Isolarion. Highly purified preparations of Leydig cells were isolated from the testes collected from adult male guinea pigs following humane sacrifice by CO, asphyxiation using the methods described by Klinefelter et al. [9] with slight modifications. Briefly, the testes were removed in situ and placed in a chilled 0.29 M isotonic sucrose solution buffered with phosphate-buffered saline, pH 7.3. Individual testicles were perfused via the testicular artery with a medium 199 (M199) dissociation buffer, decapsulated, and incubated in a shaking (90 cycles/min) water bath at 37°C. Collagenase, type 3 (Worthington Biochemical Corporation) and dispace, grade I1 (Boehringer Mannhein Corporation), were used in the testicular dissociation at a final concentration of 0.25 mg/mL each. The dissociated cell suspension was filtered through doubled 200-pm Nytex screen mesh, washed, and then elutriated at 1600 rpm in a JE-6B rotor (Beckman Instruments Inc., Palo Alto, CA) with a M199 elutriation buffer at a flow rate of 16 ml/min. Elutriated cells were washed, layered onto a 0-50% discontinuous Percoll gradient and centrifuged at 27,000g for 1 h at 4°C. The Hanks' balanced salts solution (HBSS) used to dilute the isotonic Percoll was buffered with 2.1 g/L Hepes, pH 7.2. Leydig cells were collected from the formed gradient at a density of 1.051 g/mL using density marker bead determination. The Leydig cell fraction was washed with three times the volume of a modified HBSS buffer and centrifuged at 1lOOg for 15 min to remove any residual Percoll. Leydig cells were identified by their distinctive yellow halo under phase contrast microscopy [2]. Cell viability averaging 98% was determined by the trypan blue exclusion method [17]. Cell purity, which averaged 92%, was evaluated by staining for 30-hydroxysteroid dehydrogenase [9]. Cell yield was ascertained by averaging triplicate counts with a Neubauer hemacytometer using light microscopy. Lpydig Cell Culture. Approximately 1.25 x lo6 purified Leydig cells were incubated with a maximally stimulating concentration of 100 ng/mL ovine LH in a total volume of 1.0 mL of a freshly prepared modified M199 buffer solution, pH 7.4 supplemented with sodium bicarbonate (26.2 mM), Hepes (10.1 mM), bovine serum albumin (0.1 %), L-glutamine (0.68 mM), L-asparagine (0.76 mM), penicillin-streptomycin (1 .O%), and gentamicin (0.1 %). Concentrations of sodium ascorbate ranging from 0 to 50 pM were also utilized. Incubations were performed in 1.5-mL polypropylene microcentrifuge tubes at 37°C in a shaking (120 cycles/min) water bath in duplicates. After 24 h, cells were pelleted
Oxytocin-like Peptide Production by Leydig Cells
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in a microcentrifuge at 325g for 10 min. Culture media supernatants were collected and stored at until analyzed for oxytocin by high-performance liquid chromatography (HPLC) [ 1, 71.
187
- 20 "C
HPLC Samples. One milliliter of each of the previously collected culture media supernatants was prepared for HPLC analysis by loading onto 3-mL solid phase extraction (SPE; JT Baker Chemical Company, Phillipsburg, NJ) C,, columns which were fitted with 0.45 pm nylon acrodisc syringe filters. All SPE columns were first preconditioned by washing with 1 column volume of methanol, followed by 1 column volume of distilled water. Individual supernatant samples were applied and aspirated slowly through individual SPE columns. Next, the columns were washed with 1 mL distilled water followed by 0.5 mL of a prepared 4 % acetic acid stock solution in distilled water, then air dried under vacuum for 3 min. Individual samples were eluted from the SPE columns with a 500-pL aliquot of acidified absolute ethanol (10 pL 6 N HC1/20 mL absolute ethanol). Samples were concentrated under vacuum in conical glass vials using a Savant refrigerated SpeedVac concentrator (Farmingdale, NY), then reconstituted with 50 pL ethanol and stored frozen at -20°C. Twenty microliters of each sample was injected onto the HPLC chromatography column in duplicates for oxytocin quantification. Chromatography. HPLC was performed on a Beckman 344 modular system (Beckman Instruments, Inc., San Ramon, CA) with a BAS (Bioanalytical Systems, Inc., West Lafayette, IN) L C 4 B arnperometric electrochemical detector (ECD) connected to monitor a single electrode transducer at an applied cell voltage of 0.9 V with an overall sensitivity (range) of 2 nA. HPLC was performed at a column temperature of 25 "C using System Gold software as the controller for the HPLC system. The flow rate was set at 0.5 mL/min of solvent with a premixed, vacuum-filtered (0.22-pm filter), and degassed 60% monobasic 0.15 M sodium phosphate buffer to 40% methanol blend utilized throughout (total run time = 10 min) with a 2-min column equilibration period allowed between successive chromatographic trials. The reversed phase C,, Ultrasphere ODS chromatography column (4 cm x 3 pm) and the in-line detector were preconditioned by allowing the premixed methanol buffer blend to flow through the system uninterrupted overnight prior to the application of the standards and samples the following day. Oxytocin used for preparation of the standard curve and internal standards was dissolved in acidified absolute ethanol to prepare a stock solution with a concentration of 1 mg/mL. The linear standard calibration curve generated was prepared using dilutions of the octapeptide stock solution consisting of 5000, 1000, 500, 100, and 50 ng/mL dissolved in acidified absolute ethanol. Statistics. All experiments were performed twice. Student t-test procedures were performed using Epistatistics Software [6]. p values less than 0.05 were considered significant.
RESULTS Guinea pig Leydig cells incubated at 37 "C for 24 h with a maximally stimulating dose of 100 ng/mL LH produced an oxytocin-like peptide de novo, which was identified by highperformance liquid chromatography in conjunction with amperometric electrochemical detection. Figure 1 demonstrates the similar chromatography pattern and nearly identical retention time (rt) of an oxytocin-like peptide sample (rt = 6.18 min) with that of an authentic oxytocin standard (rt = 6.22 min). A linear oxytocin standard calibration curve was calculated for the 5 multiple concentration levels injected individually. Using peak area, the least-squares straight line drawn through the 5 concentration levels generated a calibration factor (Y = 0.12X + 4.3) with a coefficient of determination greater than 95%. Unknown samples injected for oxytocin quantification were analyzed using this computer-generated oxytocin standard curve. As shown in Table 1, the addition of low concentrations (25 and 50 pM) of sodium ascorbate
M.A. Kukucka and H. P. Misra
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0.4
0.2
0
0
2
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Time ( m i n ) FIGURE 1 Chromatograms of an oxytocin standard (top) and an oxytocin-like peptide sample (bottom).
to the culture medium augmented oxytocin-like peptide production to a level of nearly twice the control values. Our findings are in accord with the results of Luck and Jungclas [ 11, 121 who reported that the addition of ascorbate to a granulosa cell culture system increased oxytocin production in vitro.
DISCUSSION Leydig cells have been utilized in vitro for a variety of functions including (1) studying the regulation of the P.,,,C,,,-hydroxylase/C,,,,,-lyase enzyme system, (2) investigating the trophic effects of LH on Leydig cell steroidogenesis, and (3) maximizing testosterone production by fully differentiated adult Leydig cells [3, 4, 81. Apart from the androgenic products, Leydig cells also synthesize other bioactive peptides that are probably involved in the paracrine and TABLE 1 Oxytocin-like Peptide Identified by HPLC Using Electrochemical Detection from Highly Purified Populations of Guinea Pig Leydig Cells Stimulated with Sodium Ascorbate Sodium Ascorbate
Oxytocin-like Peptide (Pa
(PM)
40.1' f 1.23
0 25 50
77.4' 74.2'
*
* 13.8 * 26.3
Note. Values are means SE per 1 x lo6 Leydig cells. Values with different superscript letters were significantly different @J < .05).
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autocrine regulation of testicular function. An analogous situation exists in females where the female homolog of the male testis is the ovary. In addition to the production of estrogen and progesterone, ovarian granulosa cells have been shown to manufacture and secrete oxytocin when ascorbate was added to the culture medium [ 11, 121. Embryologically, the indifferent gonad is composed of primordial germ cells which form the sex cells, and the gonadal somatic blastemal cells (i.e., non-germ cells) that form the parenchymal elements of the gonads [lo]. Developmentally, blastemal cells proliferate and differentiate into the steroid-producing cells of the indifferent gonad. Following the indifferent stages of development, ovarian blastema cells differentiate into interstitial follicular cells, which are the forerunners of the granulosa cells [lo] and possibly of the theca cells. Testicular blastema cells which do not participate in the formation of the seminiferous tubules differentiate into the interstitial cells of Leydig [ 101. Ovarian theca and granulosa cells are responsible for the production and secretion of the female steroids, while testicular Leydig cells are responsible for the production and secretion of the male androgens. The present study has attempted to demonstrate that Leydig cells produce and secrete an oxytocin-like peptide in vitro. The production of this peptide was significantly augmented when ascorbate was added to the culture medium at low concentrations (25 and 50 pM).Since Leydig and granulosa cells manufacture and secrete steroids, speculation arose that Leydig cells and granulosa cells might originate embryologically from the same cell line in the indifferent gonad. Support for this embryologic concept is presented in our current findings where Leydig cells produce an oxytocin-like peptide de novo similar to that reported for granulosa cells in vitro [11, 121. Oxytocin-like activity has now been identified in the gonads. The concentrations, while generally low, exceed blood levels and could represent tissue uptake and storage; but the fact that immunoreactive oxytocin-neurophysin I is also present in both the ovary and testis suggests that local gonadal biosynthesis of oxytocin occurs [20]. The presence, location, and biosynthesis of oxytocin in gonadal tissues suggest that it may have a wider range of physiological functions than has previously been elucidated. These results support the thesis that Leydig cells are responsible for the production of an oxytocin-like peptide which compares chromatographically with authentic oxytocin. Comparison of the amino acid composition and sequence of this oxytocin-like peptide with authentic oxytocin is already in progress to confirm whether Leydig cells possess the biosynthetic machinery for testicular oxytocin production. Acknowledgment: This research was supported in part by the Virginia-Maryland Regional College of Veterinary Medicine and by a grant from the National Institutes of Health (HL 42009). The gift of ovine luteinizing hormone (ovine LH; oLH; NIADDK-oLH-26) was kindly supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIADDK) and the National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore).
REFERENCES 1 . Bennett GW, Johnson JV, Marsden CA (1986): HPLC-ECD of neuropeptides in neural and endocrine tissues. In:
Monitoring Neurotransmitter Release During Behaviour. Joseph MH, Fillenz M, MacDonald IA, Marsden CA (Eds). Chichester, England: Ellis Horwood, pp 244-249
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M. A. Kukucka and H. P. Misra 2. Dirami G, Poulter LW, Cooke BA (1991): Separation and characterization of Leydig cells and macrophages from rat testes. J Endocrinol 130:357-365 3. Ewing LL (1989): The trophic effect of luteinizing hormone on the rat Leydig cell. J Am Coll Toxic01 8:473-485 4. Ewing LL, Wing TY, Cochran RC, Kromann N, Zirkin BR (1983): Effect of luteinizing hormone on Leydig cell structure and testosterone secretion. Endocrinology 112:1763-1769 5. Guldenaar SEF, Pickering BT (1985): Immunocytochemical evidence for the presence of oxytocin in rat testis. Cell Tissue Res 240:485-487 6. Gustafson TL (1984): Epistatistics Software. Round Rock, TX: Gustafson. 7. Johnson JV, Bennett GW, Marsden CA, Gardiner SM, Bennett T (1984): Electrochemical measurement of neurohypophyseal peptide levels in rat hypothalamic regions after adrenalectomy. Clin Exp Theor Practice A6: 19931998 8. Klinefelter GR, Ewing LL (1988): Optimizing testosterone production by purified adult rat Leydig cells in vitro. In Vitro Cell Dev Biol 24:545-549 9. Klinefelter GR, Hall PF, Ewing LL (1987): Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36:769-783 10. Latshaw WK (1987): Veterinary Developmental Anatomy. Toronto: BC Decker, pp 236-242 11. Luck MR, Jungclas B (1988): The time-course of oxytocin secretion from cultured bovine granulosa cells, stimulated by ascorbate and catecholamines. J Endocrinol 116:247-258 12. Luck MR, Jungclas B (1987): Catecholamines and ascorbic acid as stimulators of bovine ovarian oxytocin secretion. J Endocrinol 114:423-430 13. Nicholson HD, Worley RTS, Guldenaar SEF, Pickering BT (1987): Ethan-I ,2-dimethanesulphonate reduces testicular oxytocin content and seminiferous tubule movements in the rat. J Endocrinol 112:311-316 14. Nicholson HD, Swann RW, Burford GD, Wathes DC, Porter DG, Pickering BT (1984): Identification of oxytocin and vasopressin in the testis and in adrenal tissue. Regul Pept 8:141-146 15. NIH (1985): Guide for the Care and Use of Laboratory Animals. NIH publication 85-23. Bethesda, MD: National Research Council 16. NRC (1978): Nutrient requirements of domestic animals. In: Nutrient Requirements of Laboratory Animals, 3d ed. Washington, DC: National Academy of Sciences-National Research Council 17. Phillips HJ (1973): Dye exclusion tests for cell viability. In: Tissue Culture: Methods & Applications. Kruse PF Jr, Patterson MK Jr (Eds). New York: Academic Press, pp 406-408 18. Pickering BT, Ayad VJ, Birkett SD, Gilbert CL, Guldenaar SEF, Nicholson HD, Worley RTS, Wathes DC (1990): Neurohypophysial peptides in the gonads: are they real and do they have a function? Reprod Fertil Dev 2:245-262 19. Rehbein R, Hillers M, Mohr E, Ivell R, Morley S, Schmale H, Richter D (1986): The neurohypophyseal hormones vasopressin and oxytocin. Biol Chem Hoppe-Seyler 367:695-704 20. Wathes DC, Swann RW, Porter DG, Pickering BT (1986): Oxytocin as an ovarian hormone. In: Current Topics in Neuroendocrinology, Vol 6. Ganten D, Pfaff D (Eds). New York: Springer, pp 129-152 21. Wathes DC, Swann RW, Birkett SD, Porter DG, Pickering BT (1983): Characterization of oxytocin, vasopressin & neurophysin from the bovine corpus luteum. Endocrinology 113:693-698 22. Wathes DC, Swann RW (1982): Is oxytocin an ovarian hormone? Nature 297:225-227 23. Yeung WSB, Guldenaar SEF, Worley RTS, Humphrys J , Pickering BT (1988): Oxytocin in Leydig cells: an immunocytochemical study of Percall-purified cells from rat testes. Cell Tissue Res 253:463-468
HPLC DETERMINATION OF AN OXYTOCIN-LIKE PEPTIDE PRODUCED BY ISOLATED GUINEA PIG LEYDIG CELLS: STIMULATION BY ASCORBATE
M. A. KUKUCKA and H. P. MISRA Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/mL LH for 24 h in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum, and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high-performance liquid chromatography with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells 1.23, 77.4 13.8, 74.2 f stimulated with 0, 25, and 50 pM ascorbate produced and secreted 40.1 26.3 pg of an oxytocin-like peptide, respectively, per 1 x lo6 cells. These results indicate that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo and that low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro
+
+
Key Words: Oxytocin; Leydig cells; Ascorbate; HPLC; Testis; Sodium ascorbate; Peptide.
INTRODUCTION Classically, the neurohypophysial peptide hormone oxytocin has been recognized to be associated with parturition and milk ejection. In the early 1980s, the identification of a nonneural source of oxytocin secreted in significant amounts from the ovarian corpus luteum of ruminants [21, 221 rekindled a new interest in this octapeptide hormone. Shortly following the demonstration of an ovarian source of oxytocin, the presence of an oxytocin-like peptide located in the testes was reported [ 141. Using an immunocytochemical stain, approximately 80% of the intertubular (interstitial) cells, in contrast to the intratubular (seminiferous tubule) cells, immunoreacted in a manner suggesting that the interstitial Leydig cells were the source of an oxytocin-like peptide [5]. There are several indirect lines of scientific evidence that support the biosynthesis of a testicular oxytocin-like peptide de novo. Rehbein et al. [19] reported that small amounts of an oxytocin mRNA were present in rat testes. Nicholson et al. [I31 demonstrated the disappear-
From the Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. This study was presented in part at the Third Research Day Proceedings held at the Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, May 23-24, 1991. The published proceedings (No. 23919419) were catalogued with the Online Computer Library Center (OCLC) database on June 11, 1991. Address correspondence to Hara P. Misra, Professor, Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061. ARCHIVES OF ANDROLOGY 29: 185-190 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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ance of testicular oxytocin in rats following treatment with ethan- 1,2-dimethanesulfonate (EDS), which selectively depletes the Leydig cell population. Furthermore, immunoreactive oxytocin was reported to be concentrated in Leydig cell preparations isolated from dissociated rat testes [23]. Although the scientific evidence indicates that oxytocin is located in the Leydig cells, it is not certain whether a Leydig cell oxytocin-like peptide is biosynthesized de novo or is taken up and stored by the cell from some other intra- or extratesticular source [18]. In the present study, we have determined that highly purified populations of adult guinea pig Leydig cells incubated with 100 ng/mL LH produce an oxytocin-like peptide and that sodium ascorbate augments the de novo production of this peptide in vitro.
MATERIALS AND METHODS Animals. Sexually mature Dunkin-Hartley male guinea pigs weighing between 800 and 1200 g were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA). The guinea pigs were housed three to a cage in a temperature- (21 "C) and humidity- (55 %) controlled vivarium with a photoperiod of 12 h light: 12 h dark according to the Guide fur the Care and Use ofLaboratoly Animals [15]. Animals were provided a balanced NRC [16] commercially pelleted guinea pig formula diet (Agway, Inc., Syracuse, NY) and water ad libitum. Lpydig Cell Isolarion. Highly purified preparations of Leydig cells were isolated from the testes collected from adult male guinea pigs following humane sacrifice by CO, asphyxiation using the methods described by Klinefelter et al. [9] with slight modifications. Briefly, the testes were removed in situ and placed in a chilled 0.29 M isotonic sucrose solution buffered with phosphate-buffered saline, pH 7.3. Individual testicles were perfused via the testicular artery with a medium 199 (M199) dissociation buffer, decapsulated, and incubated in a shaking (90 cycles/min) water bath at 37°C. Collagenase, type 3 (Worthington Biochemical Corporation) and dispace, grade I1 (Boehringer Mannhein Corporation), were used in the testicular dissociation at a final concentration of 0.25 mg/mL each. The dissociated cell suspension was filtered through doubled 200-pm Nytex screen mesh, washed, and then elutriated at 1600 rpm in a JE-6B rotor (Beckman Instruments Inc., Palo Alto, CA) with a M199 elutriation buffer at a flow rate of 16 ml/min. Elutriated cells were washed, layered onto a 0-50% discontinuous Percoll gradient and centrifuged at 27,000g for 1 h at 4°C. The Hanks' balanced salts solution (HBSS) used to dilute the isotonic Percoll was buffered with 2.1 g/L Hepes, pH 7.2. Leydig cells were collected from the formed gradient at a density of 1.051 g/mL using density marker bead determination. The Leydig cell fraction was washed with three times the volume of a modified HBSS buffer and centrifuged at 1lOOg for 15 min to remove any residual Percoll. Leydig cells were identified by their distinctive yellow halo under phase contrast microscopy [2]. Cell viability averaging 98% was determined by the trypan blue exclusion method [17]. Cell purity, which averaged 92%, was evaluated by staining for 30-hydroxysteroid dehydrogenase [9]. Cell yield was ascertained by averaging triplicate counts with a Neubauer hemacytometer using light microscopy. Lpydig Cell Culture. Approximately 1.25 x lo6 purified Leydig cells were incubated with a maximally stimulating concentration of 100 ng/mL ovine LH in a total volume of 1.0 mL of a freshly prepared modified M199 buffer solution, pH 7.4 supplemented with sodium bicarbonate (26.2 mM), Hepes (10.1 mM), bovine serum albumin (0.1 %), L-glutamine (0.68 mM), L-asparagine (0.76 mM), penicillin-streptomycin (1 .O%), and gentamicin (0.1 %). Concentrations of sodium ascorbate ranging from 0 to 50 pM were also utilized. Incubations were performed in 1.5-mL polypropylene microcentrifuge tubes at 37°C in a shaking (120 cycles/min) water bath in duplicates. After 24 h, cells were pelleted
Oxytocin-like Peptide Production by Leydig Cells
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in a microcentrifuge at 325g for 10 min. Culture media supernatants were collected and stored at until analyzed for oxytocin by high-performance liquid chromatography (HPLC) [ 1, 71.
187
- 20 "C
HPLC Samples. One milliliter of each of the previously collected culture media supernatants was prepared for HPLC analysis by loading onto 3-mL solid phase extraction (SPE; JT Baker Chemical Company, Phillipsburg, NJ) C,, columns which were fitted with 0.45 pm nylon acrodisc syringe filters. All SPE columns were first preconditioned by washing with 1 column volume of methanol, followed by 1 column volume of distilled water. Individual supernatant samples were applied and aspirated slowly through individual SPE columns. Next, the columns were washed with 1 mL distilled water followed by 0.5 mL of a prepared 4 % acetic acid stock solution in distilled water, then air dried under vacuum for 3 min. Individual samples were eluted from the SPE columns with a 500-pL aliquot of acidified absolute ethanol (10 pL 6 N HC1/20 mL absolute ethanol). Samples were concentrated under vacuum in conical glass vials using a Savant refrigerated SpeedVac concentrator (Farmingdale, NY), then reconstituted with 50 pL ethanol and stored frozen at -20°C. Twenty microliters of each sample was injected onto the HPLC chromatography column in duplicates for oxytocin quantification. Chromatography. HPLC was performed on a Beckman 344 modular system (Beckman Instruments, Inc., San Ramon, CA) with a BAS (Bioanalytical Systems, Inc., West Lafayette, IN) L C 4 B arnperometric electrochemical detector (ECD) connected to monitor a single electrode transducer at an applied cell voltage of 0.9 V with an overall sensitivity (range) of 2 nA. HPLC was performed at a column temperature of 25 "C using System Gold software as the controller for the HPLC system. The flow rate was set at 0.5 mL/min of solvent with a premixed, vacuum-filtered (0.22-pm filter), and degassed 60% monobasic 0.15 M sodium phosphate buffer to 40% methanol blend utilized throughout (total run time = 10 min) with a 2-min column equilibration period allowed between successive chromatographic trials. The reversed phase C,, Ultrasphere ODS chromatography column (4 cm x 3 pm) and the in-line detector were preconditioned by allowing the premixed methanol buffer blend to flow through the system uninterrupted overnight prior to the application of the standards and samples the following day. Oxytocin used for preparation of the standard curve and internal standards was dissolved in acidified absolute ethanol to prepare a stock solution with a concentration of 1 mg/mL. The linear standard calibration curve generated was prepared using dilutions of the octapeptide stock solution consisting of 5000, 1000, 500, 100, and 50 ng/mL dissolved in acidified absolute ethanol. Statistics. All experiments were performed twice. Student t-test procedures were performed using Epistatistics Software [6]. p values less than 0.05 were considered significant.
RESULTS Guinea pig Leydig cells incubated at 37 "C for 24 h with a maximally stimulating dose of 100 ng/mL LH produced an oxytocin-like peptide de novo, which was identified by highperformance liquid chromatography in conjunction with amperometric electrochemical detection. Figure 1 demonstrates the similar chromatography pattern and nearly identical retention time (rt) of an oxytocin-like peptide sample (rt = 6.18 min) with that of an authentic oxytocin standard (rt = 6.22 min). A linear oxytocin standard calibration curve was calculated for the 5 multiple concentration levels injected individually. Using peak area, the least-squares straight line drawn through the 5 concentration levels generated a calibration factor (Y = 0.12X + 4.3) with a coefficient of determination greater than 95%. Unknown samples injected for oxytocin quantification were analyzed using this computer-generated oxytocin standard curve. As shown in Table 1, the addition of low concentrations (25 and 50 pM) of sodium ascorbate
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0.8
1
0.6
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0.4
0.2
0
0
2
4
6
8
Time ( m i n ) FIGURE 1 Chromatograms of an oxytocin standard (top) and an oxytocin-like peptide sample (bottom).
to the culture medium augmented oxytocin-like peptide production to a level of nearly twice the control values. Our findings are in accord with the results of Luck and Jungclas [ 11, 121 who reported that the addition of ascorbate to a granulosa cell culture system increased oxytocin production in vitro.
DISCUSSION Leydig cells have been utilized in vitro for a variety of functions including (1) studying the regulation of the P.,,,C,,,-hydroxylase/C,,,,,-lyase enzyme system, (2) investigating the trophic effects of LH on Leydig cell steroidogenesis, and (3) maximizing testosterone production by fully differentiated adult Leydig cells [3, 4, 81. Apart from the androgenic products, Leydig cells also synthesize other bioactive peptides that are probably involved in the paracrine and TABLE 1 Oxytocin-like Peptide Identified by HPLC Using Electrochemical Detection from Highly Purified Populations of Guinea Pig Leydig Cells Stimulated with Sodium Ascorbate Sodium Ascorbate
Oxytocin-like Peptide (Pa
(PM)
40.1' f 1.23
0 25 50
77.4' 74.2'
*
* 13.8 * 26.3
Note. Values are means SE per 1 x lo6 Leydig cells. Values with different superscript letters were significantly different @J < .05).
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Oxytocin-like Peptide Production by Leydig Cells
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autocrine regulation of testicular function. An analogous situation exists in females where the female homolog of the male testis is the ovary. In addition to the production of estrogen and progesterone, ovarian granulosa cells have been shown to manufacture and secrete oxytocin when ascorbate was added to the culture medium [ 11, 121. Embryologically, the indifferent gonad is composed of primordial germ cells which form the sex cells, and the gonadal somatic blastemal cells (i.e., non-germ cells) that form the parenchymal elements of the gonads [lo]. Developmentally, blastemal cells proliferate and differentiate into the steroid-producing cells of the indifferent gonad. Following the indifferent stages of development, ovarian blastema cells differentiate into interstitial follicular cells, which are the forerunners of the granulosa cells [lo] and possibly of the theca cells. Testicular blastema cells which do not participate in the formation of the seminiferous tubules differentiate into the interstitial cells of Leydig [ 101. Ovarian theca and granulosa cells are responsible for the production and secretion of the female steroids, while testicular Leydig cells are responsible for the production and secretion of the male androgens. The present study has attempted to demonstrate that Leydig cells produce and secrete an oxytocin-like peptide in vitro. The production of this peptide was significantly augmented when ascorbate was added to the culture medium at low concentrations (25 and 50 pM).Since Leydig and granulosa cells manufacture and secrete steroids, speculation arose that Leydig cells and granulosa cells might originate embryologically from the same cell line in the indifferent gonad. Support for this embryologic concept is presented in our current findings where Leydig cells produce an oxytocin-like peptide de novo similar to that reported for granulosa cells in vitro [11, 121. Oxytocin-like activity has now been identified in the gonads. The concentrations, while generally low, exceed blood levels and could represent tissue uptake and storage; but the fact that immunoreactive oxytocin-neurophysin I is also present in both the ovary and testis suggests that local gonadal biosynthesis of oxytocin occurs [20]. The presence, location, and biosynthesis of oxytocin in gonadal tissues suggest that it may have a wider range of physiological functions than has previously been elucidated. These results support the thesis that Leydig cells are responsible for the production of an oxytocin-like peptide which compares chromatographically with authentic oxytocin. Comparison of the amino acid composition and sequence of this oxytocin-like peptide with authentic oxytocin is already in progress to confirm whether Leydig cells possess the biosynthetic machinery for testicular oxytocin production. Acknowledgment: This research was supported in part by the Virginia-Maryland Regional College of Veterinary Medicine and by a grant from the National Institutes of Health (HL 42009). The gift of ovine luteinizing hormone (ovine LH; oLH; NIADDK-oLH-26) was kindly supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIADDK) and the National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore).
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