Pros~g~ndins 44:497-507,1992

THE PRODUCTION OF ARACHIDONIC ACID METABOLITES IN RAT TESTIS

G.P. Reddy, H. Prasad, S. Sailesh, Y.V.K. K,,m~r and P. Reddanna

School of Life Sciences, University of Hyderabad, Hyderabad 500 134. India

ABSTRACT

There is growing evidence that arachidonic acid is oxygenated enzymatic.ally in every cell type and that the oxygenated metabolites regulate a variety of pathological and physiological processes including reproduction. In the present study, the metabolism of arachidonic acid in the testis via cyclooxygenase and lipoxygenase pathways was analyzed. Testicular microsomes showed substantial cyclooxygenase activity as measured by the polarographic method. Analysis of the products on TLC revealed PGF2u (79.5%) as the main product followed by PGE 2 (20.3%) and PGD 2 (0.17%). At higher substrate concentrations (150 ~M), however, 6-ketoPGFl( z , the stable metabolite of prostacyclin, was observed in substantial quantities. Maximum activity of lipoxygenase was observed at pH 6.4 in both microsomes and cytosol, the activity being higher in cytosol. Analysis of lipoxygenase pathway products with arachidonic acid as the substrate, revealed the presence of 12-HPETE as the major product both in cytosol and in microsomes. Besides this, 15- and 5- HPETEs were also observed in substantial quantities.

INTRODUCTION

Arachidonic acid is the predominant polyunsaturated fatty acid found in the testis (1,2). From this, it can be presumed that it must be playing a vital role in testicular metabolism. Also, essential fatty acid deficiency was shown to result in the impairment of testicular function, resulting in male sterility (3,4). Arachidonic acid is oxygenated enzymatically by three important pathways: 1) the cyclooxygenase pathway leading to the formation of prostaglandins, thromboxanes and prostacyclins; 2) the lipoxygenase pathway leading to the formation of hydroperoxides, leukotrienes and lipoxins; and 3) the monoxygenase pathway leading to the ¢a-hydroxy and c0-carboxyfatty acids (5). Of these, the first two pathways are involved in the production of potent biologically active compounds. A number of studies are available to indicate the effects of prostaglandins on various testicular functions including steroidogenesis (6,7). However, there are limited studies with regard to the operation of lipoxygenase pathway in the testis.

Abbreviations: PG-Prostaglandin, HPETE-Hydroperoxyeicosatetraenoic acid, HETE-Hydroxyeicosatetraenoic acid

Copyright © 1992 Butterworth-Heinemann

498 Prostaglandins

Studies of Shahin et al., (8) have indicated lipoxygenase like activity in testicular microsomes. Later studies from the same laboratory have demonstrated lipoxygenase pathway in the testis (9). Their studies further indicated 15-HPETE as the major product of arachidonic acid in the ram testicular microsomes (10). The studies of Dix et al. (11), however, indicated the presence of 5- and 12-1ipoxygenases in ram tumor Leydig cells. In the present study, we report the operation of 5-, 12-and 15-1ipoxygenases in the rat testicular microsomes and cytosol.

MATERIALS AND METHODS

Adult male wistar strain rats at 100 +_ 15 days age and weighing 175 +_ 15g were employed for the present investigation.The testes were isolated from rats after cervical dislocation.The isolated testis was immediately washed in Krebs- Ringer buffer containing 5mM EDTA, to remove blood and to prevent formation of thrombi. The decapsulated testes were used for further analysis.

Estimation of cyclooxygenase (PGH synthase; EC 1.14.99.1) activity

i) Enzyme extraction Testicular tissue was homogenized in five volumes of Tris-HC1 (pH 8.0)

buffer containing 5raM EDTA (Ethylene diaminetetra acetic acid), and 5raM diethyldithiocarbamate (DDC) and centrifuged at 16,000 x g for 45 rain at 4°C. The supernatant was filtered and again centrifuged at 105,000 x g for 1hr. The microsomal pellet was rehomogenized in equal volumes of washing buffer (50raM Tris-HC1, pH 8.0 containing 100raM sodium perchlorate, 1ram EDTA and 0.1 mM DDC) and centrifuged for lhr atl05,000g. The perchlorate washed microsomal pellet was subsequently homogenized in one volume (1 mL/g wet weight) of solubilization buffer (50ram Tris-HC1, pH 8.0 containing 0.1 mM EDTA and 1% Tween 20). The Tween 20 solubilized homogenate was centrifuged at 105,000 x g for 90 min and the clear supernatant was used for enzymatic assay.

ii) Enzyme assay Prostaglandin H synthase activity was measured polarigraphically with a

Clark type of electrode on YSI model-5300 biological oxygen monitor by the method of Vander Ouderaa and Buytenhek (12). The activity of the enzyme was expressed as nmoles of oxygen consumed/min/mg protein.

Identification and quantification of the PGH synthase products

i) Incubtion conditions and extraction of the products The standard incubation mixture contained 0.075 M Tris-HCl (pH 8.0), 1.5

mM EDTA, 5 mM L-tryptophan, 0.34 mM hematin and solubilized enzyme in a total volume of 12 mL. The reaction was initiated by the addition of arachidonic

Prostaglandins 499

acid (100 ~M final concentration) containing 0.17 lxCi of (3H)arachidonic acid, and allowed to react at room temperature for 5 min and the reaction was arrested with 1N-HC1. The products were extracted by adding 24 mLof chloroform thrice and the organic phase was separated and evaporated to dryness under stream of nitrogen The residue was redissolved in 100 IxL of chloroform for TLC analysis.

ii) Separation and identificaction of products on TLC Precoated silica gel-G plates (Whatman Ltd.) were used to separate the

PGH synthase products and standards. The spotted plates were developed in a solvent system containing chloroform:methanol:acetic acid:water (100:5:1:0.8). The developed TLC plate was dried and sprayed with 50% sulfuric acid followed by heating at ll0°C for 10 min. The products were visualized under UV light and compared with known standards. Quantification was done by scraping the spots corresponding to those of the standards and measuring the radioactivity in a liquid scintillation counter.

Lipoxygenase activity Lipoxygenase activity was estimated polarographically by the method of

Grossman et al. (13) on the cytosol and microsomes prepared from rat testes as described above. The reaction was initiated by the addition of 3.66 mM linoleic acid to the reaction mixture containing 0.1 M sodium phosphate (pH 6.4) buffer containing 0.25% Tween 20 and 0.1 mL of enzyme source. The activity of the enzyme was expressed in nmoles of 02 consumed/min/mg of protein.

Identification and quantification of the lipoxygenase products The testicular cytosol or microsomes were incubated at pH 6.4 with 100

lxM arachidonic acid containing 0.1 ~tCi (3H) arachidonic acid for 5 min at room temperature. After incubation, the reaction was terminated by acidifying the reaction mixture with I N HC1. The reaction products were extracted twice with equal volumes of hexane:diethyl ether (1:1 v/v). The organic extracts were pooled, dried over anhydrous sodium sulfate, evaporated to dryness under vacuum, and dissolved in 3 mL of methanol. The extracted products were reduced with excess sodium borohydride at 4°C. After reduction, the contents were acidified and evaporated under nitrogen. The residues were redissolved in an appropriate HPLC solvent system and analyzed on straight phase HPLC (Shimadzu LC 6AD HPLC using a shimpack CLC-SLS(M) 4.6 x 250 mm column at a flow rate of I mL/min). Reaction products were separated with an HPLC solvent system of hexane: propanol:acetic acid in the ratio of 1000:15:1 v / v / v for less polar products, and 1000:28:1 v / v / v for more polar products with UV monitoring at 235nm. The various lipoxygenase products formed were identified by comparison with the retention times of the standard compounds and by co-chromatography with the standards. The various lipoxygenase standards were prepared using soybean and potato lipoxygenases as per the method described earlier (15).

500 Prostaglandins

All quantifications were based either on measurements of the radiolabel in the collected peaks or on the UV absorbance data from scanning spectrophotometry of the collected peaks.

RESULTS

Cyclooxygenase :pathway As shown in (Table 1), substantial cyclooxygenase activity was observed

in the tisticular microsomes. Analysis of the products from arachidonic acid on TLC (Fig. 1) revealed that PGF2( z was the predominant prostaglandin (79.5%), followed by PGE 2 (20.3%) and then PGD 2 (0.17%). Besides these PGs, there was a peak corresponding to 6-ketoPGFlo ~ the breakdown product of prostacyclin, when the arachidonic acid concentration was increased to 150 ~M (Fig. 1).

Lipoxygenase pathway Lipoxygenase activity was recorded both in testicular cytosol and

microsomes, the activity being higher in the cytosol (Table I). The pH optimum for both fractions was 6.4 (Fig. 2). The product profile of cytosolic lipoxygenase with arachidonic acid obtained by HPLC with the less polar solvent is presented in Fig. 3. The products obtained were collected, scanned for a typical conjugated diene spectrum and then compared with standard HETEs. As shown in the HPLC chromatogram, two peaks with retention times of 9.25 and 16.43 min showed a typical conjugated diene spectrum with radioactivity in the peaks. These two peaks had retention times similar to the standards for 12-HETE andlS-HETE. The peak with a retention time of 9.25 was collected, evaporated and co-injected with standard 12-HETE. As shown in the co-chromatogram (inset A of Fig. 3), a single peak was observed with a retention time of 9.50 indicating that the original 9.25 peak was 12-HETE. Similarly, the peak with an original retention time of 16.43 was co-chromatographed with the 15-HETE standard. As shown in the co-chromatogram (inset B of Fig. 3), a single peak with a retention time of 16.57 was observed. This suggests that the original peak was 15-HETE. Further quantification of the products was done based on radioactivity measured in these samples.

When the cytosolic lipoxygenase products were separated on HPLC using a more polar solvent (H:P:A; 1000:22:1 at a flow rate l m L / m i n ), a product with a retention time of 32.57 (Fig.4) was obtained with a chracteristic conjugated diene spectrum and radioactivity. This compound had a retention time similar to the authentic 5-HETE. When the peak with a retention time of 32.57was co- injected with the 5-HETE standard , they co-eluted as a single peak with a retention time of 36.38 (inset of Fig. 4). Thus, the cytosolic fraction produced 12- HETE as the major lipoxygenase product with 15- and 5-HETEs as the minor products in the ratio of 49.5 : 37.3 : 13.2.

Prostaglandins 501

Figure 1. Separation of tesficular microsomal prostaglandins on TLC.

lane 1. lane 2. lane 3.

o lane 4.

lane 5.

Standard TXB 2 Standard 6-keto PGFlc t Standards A. = PGF2c u C. = PGE2, D .= PGD 2 Prostaglandins formed when microsomes are incubated with 100 ~M arachidonic acid, E. = lipoxygenase products, and F. = arachidonic acid. Prostaglandins formed when microsomes are incubated with 150 ~tM arachidonic acid.

Table 1. Relative Distribution of Arachidonate Cyclooxygenase and Lipoxygenase Activities in Rat Testicular Cytosol and Microsomal Fractions.

Lipoxygenase Cyclooxygenase activity activity

Cytosol Microsomes

Specific Activity* 39.75 21.95

*n moles of 02 consumed/min /mg protein

Microsomes

6.5

502

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Prostaglandins

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7 B

ronge F i g t ~ 2. Effect of pH on the ac t iv i ty l e w i s of testieular lipoxygerm~e.

Figure 3. HPLC SeparaU.orj of t ea t icu~r ¢ytoaotic lipoxygena,a products from ineubet|on with H-aro~d~lo~e acid. l n ~ A and B ~aow co- c h r o m a t o g r a m s of the 9.25 peak + 12-HETE and the 16.43 peak + 15-HETE respectively.

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Prostaglandins 503

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4. HPLC Seporatlon of reticular c~..8oI|¢ lipoxygena~ products from btcubatlon with FI-amchldonie acid.

F~ure S.

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504 Prostaglandins

Similarly the microsomal lipoxygenase products after reduction were extracted, and then separated on HPLC straight phase using solvent system 1000:28:1 (H:P:A) at flow rate lmL/min. As indicated in the chromatogram (Fig. 5) all the 3 HETEs were identified as 12-, 15- and 5-HETE based on the spectrophotometric and radioactive studies along with co-chromatography.

DISCUSSION

The male gonad is a complex organ that requires many hormones and nutritional factors for normal activity. Many nutritional factors are known to affect testicular function.Among the nutritional factors, the lipids especially the essential fatty acids play an important role in the testicular function(3). The essential fatty acid deficiency was shown to result in the impairment of testicular function, resulting in male sterility(4). Testicular tissue has an unusually high concentration of polyenoic fatty acids such as arachidonic acid(I,2). It is well known that arachidonic acid is the main precursor of a number of bioactive compounds collectively called eicosanoids (6,7), which are formed predominantly by two distinct enzymatic pathways. Any number of these products might play an important role in the regulation of testicular function.

In the present study the cyclooxygenase activity as measured by polarographic technique, was detected in the microsomes of rat testis. Further analysis of the metabolites formed indicated that PGF2a is the most predominant prostaglandin formed, followed by PGE 2 and PGD 2 . Besides these, 6-keto PGFla also was produced in major quantities in the testis at higher substrate concentration. Studies of Shahin et al (11) also indicated the presence of PGF2a, PGE 2 and 6-Keto PGFla in ram testis. In light of the fact that testicular tissue has an unusually high concentration of polyenoic fatty acids, such as arachidonic acid, it can be conceived that AA could be oxidized through the lipoxygenase pathway in testis, aimed at the regulation of testicular functions. In this direction, there is practically no information on the metabolism of arachidonic acid via the lipoxygenase pathway except for the pioneering work of Grossman and his group from Israel. They first identified the lipoxygenase- like enzyme in rat testicular microsomes (8). The enzyme was shown to metabolize linoleic acid into 13-hydroxy, and 9-hydroxy linoleic acid in the ratio of 67:13 (9) . They also identified a major product as 13-hydroxy - octadecatrienoic acid. Their later studies with arachidonic acid revealed that 15- HPETE and 15-HETE as the products of testicular microsomal lipoxygenase pathway, suggesting the presence of 15-1ipoxygenase in the testis(5). Dix et al. (11) identified the presence of 5-HETE andl2-HETE in ram tumor Leydig cells.

In the present study, lipoxygenase activity was detected both in the cytosol as well as in microsomes of the rat testis. The activity recorded was greater in the cytosol than that in the microsomes. The cytosol as well as microsomal lipoxygenases showed optimum activity at pH 6.4. Shahin et al. (8), on the other hand, observed pH optimum at 6.0. This increase in optimum pH could be due to the use of sodium phosphate buffer in the present study, which

Prostaglandins 505

causes a shift in the optimum pH towards 6.5(8). The testicular lipoxygenase activity with arachidonic acid as the substrate was substantial and can be measured both oxygraphically as well as on spectrophotometer. The significant finding of the present study is the presence of cytosolic lipoxygenase in the testis. In fact most of the lipoxygenases,from animal as well as plant sources are cytosolic (16-19). Only a few membrane bound lipoxygenases were reported like nuclear membrane bound lipoxygenase of tulip bulbs (20).Thus testicular tissue seems to be unique in having the lipoxygenase activity in the cytosol as well as in the microsomes. The major product of lipoxygenase pathway reported with arachidonic acid as the substrate was 15-HETE in the testicular microsomes (10), suggesting the presence of 15-1ipoxygenase in the testis. In the present study, both in cytosol as well as in microsomes, 12-HETE is the major product, suggesting the operation of 12-1ipoxygenase pathway in the testis. This is contradictory to the studies of Shahin et al., (10) where they have observed no traces of 12-HETE in testicular microsomes.This discrepancy could be due to the lack of resolution of 12-&IS- H(P)ETEs in the techniques employed. The TLC method employed by Shahin et al. (10) may not be able to resolve 12 & 15- I-IPETEs.Besides the separation of lipoxygenase products on HPLC using reverse phase is not the ideal method. The ideal method for the separation of 12 and 15- H(P)ETEs would be a straight phase silica column. Further, to obtain the best separation of 12 andl5-H(P)ETEs on silica column, the I-IPLC solvent of less polarity can be employed. As shown in the present study, the 12-and 15-H(P)ETEs can be separated very well by using the solvent system of H:P:A (1000:15:1) without any possibility for erroneous conclusions. Thus,the present study demonstrates the presence of the enzyme 12-1ipoxygenase in both testicular cytosol and microsomes. Similarly, the formation of 12- and5-HETEs in leydig cells of a ram tumor were reported using TLC analysis of the products (11). Besides 12-HETE, 15-HETE also was observed as the minor product of lipoxygenase pathway in the testis cytosol and microsomes. This observation coincides with that of Shahin et al (10), where 15- H(P)ETE was observed in microsomes of the testis. Another significant feature of the present study is the formation of 5-HETE also in the cytosol and microsomes. The presence of 5 - ~ as the product of lipoxygenase in the testis could not be detected on straight phase with solvent system H:P:A (1000:15:1). A more polar solvent system with H:P:A (1000:22:1) had to be employed to identify the presence of 5-HETE in the products of AA in the testis. This study reveals the operation of 5-1ipoxygenase pathway in the testis, which is responsible for the production of physiologically active compounds like leukotrienes, lipoxins, hepoxylins and trioxylins. However, in the present study no other polar products, similar to leukotrienes were identified in the testis. Studies of Dix et al (11) have indicated the production of LTB 4 in ram tumor Leydig cells by RIA technique. However, a number of people employed leukotrienes and studied their effects in various testicular functions (21). The lipoxygenase products also were shown to regulate the basal and LH-stimulated testicular androgen biosynthesis (22-24). Besides the gonadotropins, the intra testicular factors regulating steroidogenesis

506 Prostaglandins

could be the eicosanoids(24). However, detailed studies are required in the production and effects of eicosanoids during gonadotropin -stimulated steroidogenesis.

The present study reveals the production of 12-HPETE,15-HPETE & 5- HPETE in the rat testis. However it is not clear whether all these compounds are the products of a single lipoxygenase like the potato enzyme (15) or the products of three independent enzymes. Further studies on purification and characterization of testicular lipoxygenase(s), especially the amino acid sequence at the active site (25) which determines positional specificity ,are required to unravel the mechanism involved in the production of all three HPETEs.

ACKNOWLEDGEMENTS

These studies were supported by The Department of Biotechnology grants (BT/TF/03/25/007/89). Panduranga Reddy is the Senior Research Fellow of the Council of Scientific and Industrial Research.

REFERENCES

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10. Shahin, I., M. Soser, M. Cojacaru, and S. Grossman. Arachidonic acid metabolism by perfused ram testis. Int. J. Biochem. 21: 7.

Prostaglandins 507

11. Dix, J.C., A.D. Habberfield, M.H.F. Sullivan, and B.A. Cooke. Inhibition of steroid production in Leydig cells by non-steroidal anti-inflammatory and related compounds: evidence for the involvement of lipoxygenase products in steroidogenesis. Biochem. J. 219:529. 1984.

12. Vander Ouderaa, F.J.G. and M. Buytenhek. Purification of PGH Synthase from sheep seminal vesicular glands. Methods. Enzymol. 86:60. 1982.

13. Grossman, S., A. Ben-Aziz, P. Bodowski., I. Ascarelli., A. Gertler, Y. Birk, and A. Bondi. Enzymatic oxidation of' carotene and linoleate by alfalfa: Extraction and separation of active fractions. Phytocl~vn 8:2287. 1969.

14. Ben-Aziz, A., S. Grossman, I. Ascarelli, and P. Budowski. Linoleate oxidation induced by lipoxygenase and heme proteins: direct spectrophotometric assay. Anal. Biochem. 34:88. 1970.

15. Reddanna, P., J. Whelan, K.R. Maddipati, and C. Channa Reddy. Purification of arachidonic acid 5-1ipoxygenase from potato tubers. Methods. Enzymol. 187:268. 1990.

16. Furukawa, M., T. Yoshimoto, K. Ochi, and S. Yamamoto. Studies on arachidonate S-lipoxygenase of rat basophilic leukemia cells. Biochem. Biophys. Acta 795:458. 1984.

17. Yoshimoto, T., M. Yamoto, K. Ochi, and S. Yamamoto. Arachidonate 12- lipoxygenase of porcine leukocyte with activity for 5-hydroxyeicosa- tetraenoic acid. Biochem. Biophys. Acta 71__~3:638. 1982.

18. Shinjo, F., T. Yoshimoto, C. Yokoyama, S. Yamamoto, S. Izumi, N. Komatsu, and K. Watanabe. Studies on porcine arachidonate 12- lipoxygenase using its monoclonal antibodies. J. Biol. Chem. 261:3377. 1986.

19. Van Den, T. and E.M.T. Mendoza. Purification and characterization of two lipoxygenase isoenzymes from cowpea [Vigna unguiculata (L) Walp]. J. Agrl. Food. Chem. 30:54. 1982.

20. Reddanna, P., J. Whelan, P. S. Reddy, and C. Channa Reddy. Isolation and characterization of S-lipoxygenase from tulip bulbs. Biochem. Biophys. Res. Commun. 157:1348. 1988.

21. Abayasekara, D.R.E., L. O. Kurlok, J. Y. Jeremy, P. Dandara, R. M. Sharper, and B. A. Cooke. The levels and possible involvement of leukotriene B and prostaglandin F in the control of interstitial fluid volume in the rat testis. Int. J. Androl. 13:408. 1990.

22. Abayasekara, D.R.E. Leukotrienes and prostaglandins in testis function. In: Int. Sym. on "Recent advances in male reproduction", Hyderabad, India, 1988.

23. Pandu Ranga Reddy, G., M. Prasad, and P. Reddanna. Stimulation of testicular androgenesis by lipoxygenase products of arachidonic acid. In: 8th Int . Sym. on " Prostaglandins,. Leukotrienes, Lipoxins & PAF XI Washington Int.Spring. Sym. Washington DC, U.S.A. 1990.

24 Pandu Ranga Reddy, G., M. Prasad, S. Sailesh, Y. V. Kiran Kumar, and P. Reddanna. Arachidonic acid metabolites as intratesticular factors controlling androgen production. Int. ]. Androl. in press. 1992.

25. Sloane, D.L., R. Leung, C. S. Craiks, and E. Sigal. A primary determinant for lipoxygenase positional specificity. Nature 354:149. 1991.

Editor: C. Weems Received: 2-27-92 Accepted: 8-4-92