Prostaglandins E2 and F2α decrease plasma testosterone levels in male rats

PROSTAGLANDINS E 2 AND F2~ DECREASE PLASMA TESTOSTERONE LEVELS IN MALE RATS.

S.K. Saksena, S. El Safoury and A. Bartke

Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545

Abstract Prostaglandins PGE 2 and PGF2a depressed significantly the plasma testosterone levels when given subcutaneously to mature male rats at a dose of 500 vgm/rat~njection, b.i.d, for three days in normal saline or 250 ~gm/rat/injection, b.i.d., for four days in sesame oil. Comparable treatments of PGE 1 and PGFla (250 vgm) in sesame oil or lower doses of PGE 2 and PGF2a in saline (25 vgm) failed to depress significantly the plasma testosterone concentration. In addition, the 500 vgm treatment of PGE 2 and PGF2a lowered plasma LH concentration although this decrease was significant only with PGF2a. Constriction of testicular vasculature and~or a decrease in plasma LH levels may account for, or contribute to this action of the prostaglaT~dins.

Acknowledgements Our thanks are due to Dr. J. Pike of Upjohn Company for the generous supply of prostaglandins; to Carol Roberson for carrying out LH determinations, to Dr. B.V. Caldwell for the testosterone antiserum; to Dr. G.D. Niswender and Dr. A.R. Midgley for LH antiserz~, Dr. L.E. Reichert, Jr. for LH Standards. This study was supported by Ford Foundation through its Training Program in Physiology of Reproduction (SKS & S. El S) and NIB grant 1K04 HD70369-01.

Accepted June 20, 1973

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INTRODUCTION It has been established that ¢idministration of prostaglandin F2a can terminate eorpllS luteLim function in most of the subprimate speeic~ (l). The studies in sheep (2~3) indicate that prostaglandins are probably the naturally occurring luteolytie factor. Other physiologic{~l roles of prostaglandins in the reproductive fl,lctions of m~m~nals have also been proposed (4-7) however, very little information on the affects of PGs on male reproductive functions is available. Recently, a decrease of plasma testosterone levels in male mice after PGF~ administration was reported (8). In an unpublished experlment (Saksena, Lau and Bartke) a significant decrease was observed in serLun testosterone levels after a~ninlstration of PGF~L ( ~ and other PGs • in mice. In addition subcutaneous injections of PGE 1 and PGE 2 in male rats inhibits spermatogenesis and results in depression of weight of some of the accessory reproductive glands (9). In view of these findings we decided to examine the effects of PG administration on plasma testosterone levels~ plasma LH levels, the weight of the testes and of the seminal vesicles in PGtreated male rats. MATERIALS AND METHODS Experiment i: Charles River CD R rats of proven fertility weighing between 350-400 g were used. Prostaglandin E 2 and F 2 were dissolved in sterile saline. Fresh solutions were made before each experiment and refrigerated during the treatments. Injections were given subcutaneously in 0.4 ml at 0900 and 2100 h daily for three days at a dose level of 25 or 500 ~g/injeetion. Three hours after the last injeetion~ animals were anesthetized with Nembutal. The body cavity was then opened and blood samples were collected directly from abdominal aorta in i0 ml heparinized syringes. Samples were immediately centrifuged and the peripheral plasma obtained was frozen and stored at below -15°C until assayed. Immediately thereafter, seminal vesicles (with coagulating gland) and testes were removed~ blotted in two folds of filter paper and weighed.

Experiment 2: Adult male rats of the Charles River CD R s t r a i n , weighing 250-300 g were used. APi)ropriate amounts of PGs (PGEI, PGE23 PGFI~ and PGF2(~) were dissolved in sesame oil and were kept refrigerated between injections. PGs were administered subcutaneously in a volume of 0.2 ml every 12 hours for four days at a dose level of 250 ~g/injection. The animals were decapitated 12 hours after the last injection and the blood was collected in heparinized centrifuge tubes. The blood obtained was centrifuged within 5 minutes and the plasma obtained was stored at -15°C until assayed.

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All experimental animals were kept in a temperature and illumination controlled room (].4 h l i g h t : 10 h d a r k ) a n d f e d Agway Charles River Formula and allowed water ad libitum. Control animals in both experiments received equivalent amounts of the respective vehicles subcutaneously. Prostaglandins used in the study were in solution in normal saline or in sesame oil.

flssay for Testosterone

(T):

Plasma testosterone concentration was determined by radioimmunoassay (10). The ether extract of plasma was chromatographed on Sephadex-LH-20 columns using 232,4-tr~lethylpentane:methanol: benzene (90:5:5) as a solvent system. The sensitivity of the assay was approximately 50 pg T and the values of water blank were below 10 pg T. Assay for Luteinizin~ Hormone: Concentration of LH in the peripheral plasma was measured by radioimmunoassay (RIA) with minor modifications of the technique described by Niswender et al. (ii). Iodination was performed by the technique of Greenwood et al. (12) as described by Scaramuzzi et al. (13). The LH standard (LER-1213A) as determined by RIA, was 0.049 times as potent as HIH-LH-512. Plasma LH concentrations were measured in 400 pl aliquots in duplicate and all LH measurements were conducted in the same assay. The results were analyzed by analysis of variance and Duncan's test. RESULTS The results of the first experiment where male rats of proven fertility were treated with 25 or 500 pg of PGE 2 or PGF2~ in saline b.i.d, for three days are depicted in Table i. IT can be seen that both the PGs at a low dose (25 ~g) were quite uneffective in altering the plasma testosterone or luteinizing hormone concentrations. However; the treatment with 500 pg/injection caused a significant depression in testosterone concentration apparently more marked with PGE 2. In contrast~ plasma LH level was significantly decreased by PGF2~ J while the apparent effect of PGE 2 was not significant. There was no effect of PGF 2 and PGE 2 on seminal vesicles weight. Significant decrease in the weight of testes was noticed in the animals treated with PGE 2 (Table 2). In experiment 2~ mature male rats were given 8 injections b.i.d, of PGs (250 p~/injection) in sesame oil, and the results are shown in Table 3. In this experiment the PGE 1 and PGF 1 had no effect on plasma testosterone levels, but both PGE 2 and ~GF 2 caused a siguJficant drop in tile testosterone concentration. T~e decrease with PGE 2 seemed more marked.

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Table 2 Effects of six subcutaneous injections of 500 ~g of PGE 2 or PGF2a on testes and seminal vesicles weight in rags

Treatment

Mean body weight (Mean ~ SE) g

Weight of testes mg/100 g body weight (Mean ~ SE)

Saline (control)

333 ~ 13.7

926.5 i 20.9 (i0)

133.2 * 3.7 (I0)

PGE 2

343 ~ 11.6

837.2 • 26.6 (10)

144.7 * 4.6 (i0)

PGF2~

355 i

898.9 ~ 26.8 (12)

131.6 ± 5.0 (12)

8.0

Weight of seminal vesicles mg/100 g body weight (Mean i SE)

Figures in parenthesis shows the number of animals used. . Indicates that the treatment was significantly different from the respective control at P < 0.05 (t - test).

Table 3 Plasma testosterone levels in rats treated with prostaglandins EI~ E23 Flff and F2~ in sesame oil at a dose level of 250 ~g/injection for 4 days Treatment

Time of sample collection

Seasme oil (control s)

Testosterone levels ng/ml Mean i SE 2.34 i 0.28 (9)

PGE 1

12 h after the

1.48 i 0.2 (8)

PGE 2

last injection

1.03 i 0.22 (i0)

PGFIc~

1.76 i 0.39

(9)

PGF2~

1.39 i 0.22 (12)

**Different from oil injected controls; P < 0.05. Treatments were given as subcutaneous i n j e c t i o n s . F i g u r e s i n p a r e n t h e s i s show tlle number of animals used.

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DISCUSSION

The results of this study demonstrate that administration of PGE 2 and PGF2~ to adult male rats can cause significant decrease in plasma testosterone levels. This effect of PGs did not seem to depend on differences in dose, vehicle; age or interval between last injection and collecting the blood. The present findings are consistent with observations that treatment with PG~2~ decreases plasma testosterone levels and inereases concentration of esterified cholesterol in the testes of mice probably resulting from the inhibition of testieular steroidogenesis (8) and that chronic treatment with PGE 1 and E 2 inhibits spermatogenesis and decreases the weight of testes and accessory reproduetive glands in rats (9). In our rats seminal vesicles weight was not reduced by PG administration; probably because of the short duration of the treatment (3 days vs 15 days in the work of Erieksson (9)). The apparent decrease in plasma testosterone levels in rats given PGE 1 or PGF- was not significant but neverthe less suggests that tNese PGs±~iven in sufficient doses, could also reduce testosterone levels. PGE 2 and PGF2~ were shown to decrease testes blood flow in acute experiments on anesthetized rats (14). This may suggest that the decrease of plasma testosterone levels after PG administration is due to reduced blood supply of the testes. However; this explanation is not supported here since plasma LH levels were decreased in PG treated rats (Table i). Reduction of testosterone production due to direct effect of PG on the testes would be expected to cause an increase in release of pituitary LH. Perhaps administration of PG resulted in decreased LH release and the fall in plasma T levels was secondary to this effect. Other possibility could be that PGE 2 and PGF2_ may act simultaneously both on the pituitary to lower LH secretion and on the testes to inhibit testosterone secretion. Such explanation is consistent with out data but does not agree with the observation that PG causes release of pituitary LH in female rats (15) and sheep (16). Since the effect of PGF2~ in the release of LH from the sheep pituitary depends on the endocrine state of the animal; it seems possible that PGs have different effects on LH release in males than in females. In conclusion, we presented evidence that administration of high doses of PGE~ and PGF~ deereases plasma testosterone levels in male rats. Constriction of the testicular vaseulature and/or the decrease in plasma LI{ levels may account for or contribute to this action of prostaglandins in the males.

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REFERENCES i.

Phurriss, B.B.~ S.A. Tillson and R.R. Erickson. Prostaglandins in reproductive physiology. Recent Progr. Hormone Res. 28, 51 (1972).

2.

McCracken, J.A., D.T. Baird and J.R. Goding. Factors affecting the secretion of steroids from the transplanted ovary in sheep. Recent Progr. Hormone Res. 27~ 537 (1971).

3.

McCracken~ J.A. Prostaglandin F2~ and corpus luteum regression. Annals N.Y. Acad. Sci. 180, 456 (1971).

4,

Speroff~ L. and P.W. Ramwell. Prostaglandins in reproductive physiology. Am. J. Obst. Gynec. 107, Iiii (1970).

5.

Aiken~ J.W. Aspirin and indomethacin prolong parturation in rat: Evidence that prostaglandins contribute to its expulsion of fetus. Nature 240, 21 (1972).

6.

Armstrong, D.T. and D.L. Grinwich. Blockade of spontaneous and LH induced ovulation in rats by indomethaein, an inhibitor of prostaglandin biosynthesis. Prostaglandins ~, 21 (1972).

7.

Chester~ R., M. Dukes, S.R. Slater and A.L. Walpole. Delay of parturation in the rat by anti-inflammatory agents which inhibit the biosynthesis of prostaglandins. Nature 240, 37

(1972). 8.

Bartke, A.~ N. Musto, B.V. Caldwell and H.R. Behrman. Effect of cholesterol esterase inhibitor and of prostaglsndin F2~ on testis cholesterol and on plasma testosterone in mice. Prostaglandins ~, 97 (1973).

9.

Ericeson~ R.J. Prostaglandins (E1 and E2) and reproduction in the male rat. Adv. Bioseiences, Pergamon Press/Vieweg 9~

737 (1973). 10.

Bartke~ A.~ R.E. Stelle, N. Musto and B.V. Caldwell. Fluctuations in plasma testosterone levels in adult male rats and mice. Endocrinology 92~ 1223 (1973).

Ii.

Niswender~ G.D.~ A.R. Midgley~ S.E. Monroe and L.E. Reichert, Jr. Radioimmunoassay of rat luteinizing hormone with antiovine LII serum and ovine LH-131I. Proc. Soc. Exp. Biol. Med. 128, 807 (1968).

12.

Greenwood, F.C.~ W.M. Hunter and J.S. Glover. The preparation of 131I-labelled human growth hormone of high specific radioactivity. Bioehem. J. 89, 114 (1952).

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13.

Searamuzzi, R.J., B.V. Caldwell and R.M. Moor. Radioimmunoassay of Lll and estrogen during the estroue cycle of the ewe. Biol. Reprod. ~, 110 (1970).

14.

Free, M.J. and R.A. Jaffe. Effect of prostaglandins on blood flow and pressure in the conscious rat. Prostaglandins ~3

483 (1972). 15.

Labhsetwar, A.P. Effects of prostaglandin F 2 on pituitary luteinizing hormone content of pregnant rats. A possible explanation for the luteolytic effect. J. Reprod. Fert. 23,

155 (1970). 16.

242

Carlson, J.A.~ B. Barcikowski and J.A. McCraeken. Prostaglandin F, and the release of LH in the sheep. J. Reprod. Fert. (in press).

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