Testosterone stimulates prostanoid production by rat vas deferens

Prostaglandins Leukotnenes and Essential Fatty Acids (1992) 46. 31 l-311 0 Longman Group UK Ltd 1992

Testosterone Stimulates Prostanoid Production by Rat Vas Deferens H. A. Peredo and E. S. Borda Centro de Estudios FarmacoMgicos y Bothnicos (CEFYBO), Consejo National de Investigaciones Cientificas y Te’cnicas de la Reptiblica Argentina (CONICET) (Reprint requests to HAP’) ABSTRACT.

The rat isolated vas deferens produces and releases prostanoids into an incubation medium. Production of these substances from the exogenous precursor 14C arachidonic acid was studied in prepubertal, pubertal and adult animals. Synthesis of prostaglandin F, prostaglandin E, prostaglandin D and thromboxane B2 is lower in prepubertals arid increases significantly in pubertals, with no further modifications in adults. Castration of pubertals and adults dramatically reduces the production of all measured arachidonic acid metabolites but does not modify it in prepubertals. Replacement therapy with testosterone propionate significantly enhances prostanoid production in pubertal and adult castrated rats. Similar treatment on normal prepubertals also increases synthesis, indicating that androgens could be modulators of prostanoid synthesis in vas deferens. The lower effects obtained treating castrated adults with progesterone and with 17-p estradiol suggest an action, at least partially specific for androgenic steroids. It is concluded that prostanoid production by the rat vas deferens from an exogenous predursor is closely related to the presence of androgens.

INTRODUCTION

MATERIALS AND METHODS

The vas deferens of rats and mice has significantly higher values of prostanoid (arachidonic acid (AA) metabolites via cyclooxygenase) synthesis than any of the accesory sex organs studied (l-5). There is evidence that androgens, which are an important factor in the regulation of puberty and in the sexual maturation process, increase abruptly its serum levels in male rats at approximately 30 days of age (6). In addition, there have been reports of interactions of androgens and prostanoids in rat plasma (7), male reproductive system of rats (5, 8) and mice (1) and in perfused ram testis (9). Moreover, it has been showed that prostaglandins (PG) undergo age-dependent changes in rat vas deferens and epididymis (4, 5). The aim of the present study is to investigate the influence of age and steroid hormones on the prostanoid production by isolated rat vas deferens from an exogenous substrate.

Wistar male rats were divided in three groups of different age: prepubertal (30 days), pubertal (60 days) and adult (90 days). In each group, 10 animals were used as controls, and 12 were castrated and killed 30 days later; 6 castrated animals in each group and 6 normal prepubertals were injected with testosterone propionate; 6 adult castrated rats were injected with 17-p estradiol and 6 with progesterone. Finally, 5 adult castrated were injected with vehicle (sesame oil) only. In each case of testosterone, progesterone or vehicle treatment, animals were injected intraperitoneally for 10 days beginning in the 20th day post castration and killed 24 h after the last dose. Testosterone propionate dose was 6.6 mg/kg in 0.5 ml sesame oil. Progesterone dose was 6.5 mg/kg in the same vehicle. Animals treated with estradiol received 0.5 ug injected intraperitoneally in the 28th day post castration and 1 ug the following day. and were killed 24 h later. Animals were killed by cervical dislocation. and immediately both vas deferens were removed and placed in a Petri dish containing Krebs-Ringer-bicarbonate solution (KRB) gassed with 5% CO?-95% O1 (carbogen) at room temperature. Tissues were dissected out of the

I Correspondence to: Horatio A. Peredo, ININFA, Junin 956, ( I I 13)Buenos Aires, Argentina. Date received 8 November I99 1 Date accepted 23 December 199 I 311

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surrounding tissue (adipose tissue, blood vessels and connective sheath), and washed intraluminally twice by flushing with 1 ml KRB. The washed tissues were cut into pieces (approximately 1 mm long), placed in flasks containing 2 ul (1-14C) AA (53.9 mCi/mmol), in 2 ml KRB and incubated in a Dubnoff thermostatic bath at 37 “C under atmosphere of carbogen with continuous shaking. After 120 mitt, incubation was stopped by addition of 0.125 ml 1 N citric acid to the medium. Acidified media were treated for prostanoid extraction three times with 1 volume of chloroform. Pooled chloroformic extracts were dried under a nitrogen stream, resuspended in 0.2 ml chloroform/methanol 2 : 1 (V/V) and applied to thin layer chromatography plates (TLC plastic precoated sheets, silica gel G, Merck). The plates were developed twice in the solvent system bencene: dioxane : acetic acid (60 : 30 : 3, V/V). Plates with authentic PGEZ, PGF2,, PGDl and thromboxane B2 (TXBJ were run in paralell. The position of these standards were visualized by spraying the dried plates with a 10% solution of phosphomolybdic acid in ethanol, followed by heating at 70 “C for 10 min. The TLC plate was cut in sections of 1 cm and its radioactivity measured by liquid scintillation counting. Radioactivity in each section was divided by the total radioactivity of the whole plate, and expressed as a percentage of the total. ‘“C-AA metabolites were identified by comparison with the standards run in parallel. All values are given as means + SEM. Results were considered significant if p
RESULTS Figure 1 shows the basal conversion by isolated vas deferens of (1 J4C) AA into four metabolites: PGE, PGF, PGD2 and TXB2, in prepubertal, pubertal and adult rats. The percentage of the four metabolites was lower in the first group, with a significant increase in the second, and no further significant modifications at 90 days of age (p: prepubertal vs pubertal: ~0.01, ~0.05, co.01 and co.01 for PGF, PGE, PGD, and TXB2 respectively). The possible influence of the seric androgen concentration on vas deferens prostanoid production was studied using castrated animals, considering that castration dramatically reduces those concentrations. Bilateral castration reduced 14C-AA conversion in both pubertal (Fig. 2A) and adult animals (Fig. 2B) (p
Prostanoid Production Basal Percent of AA Conversion

PGF

TxB,

PGE

PGD

Prostanoids m

Prepubertal

m

Pubertal

Adult

Fig. 1 Basal conversion of (I -?I) arachidonic acid (AA) in the vas deferens from prepubertal(30 days), pubertal (60 days) and adult (90 days) rats, expressed as % of conversion and represented by means f SEM; n = 5. Significantly different (*: ~~0.05: **: p
PGE, and TXB,; co.05 for PGD2; adult testosterone treated vs untreated: co.01 for PGF, PGE and TXB*, co.05 for PGD2.) The same treatment, administered to intact prepubertal rats, also resulted in an increase of the synthesis (Fig. 3) (p
DISCUSSION The present results show that the isolated rat vas deferens synthesizes prostanoids from an exogenous precursor and releases these metabolites to an incubation medium. The amount of prostanoids produced is different in prepubertal animals compared to pubertal and adults. This increase in prostanoid synthesis with age is

Testosterone

Stimulates Prostanoid

Pubertal Castrated Percent

Percent

Control

Castrated

PGE

TxE2

PGD

Prostanoids

Prostanoids m

3 I3

of AA Conversion

PGF

PGD

TxB2

PGE

by Rat Vas Deferens

Adult Castrated

of AA Conversion

PGF

Production

m

Caat+Toatoat

m

Control

Castrated

m

Cast+Test

B Fig. 2 Effects of castration and testosterone propionate treatment on conversion of (I-‘%) AA in the vas deferens from pubertal (A) and adult (B) rats. Significantly different (*: p
in agreement with results reported by Gerozissis and Dray for PGE, (4). The onset of puberty in the male rat (10) is associated with a dramatic increase of androgenic hormones serum levels (1 l), simultaneously with an increment of prostanoid production by the vas deferens. So, we decided to study prostanoid synthesis in castrated prepubertal, pubertal and adult animals, a condition associated with dramatically decreased androgen levels (12). Thus, we observed that in such groups with elevated levels of prostanoid production (prepubertal and adult), castration significantly reduced that production, as previously described for PGE? and PGF2 in rats (5) and mice (1). On the other hand. conversion of 14C-AA to Table Conversion of “C arachidonic from castrated rats Treatment

None 17-p Estradiol Progesterone

acid (AA) in the vas deferens

Percent of conversion PGF PGE 1.0+0.1 1.9kO.l 1.9 t 0.2

1.3 f 0. I 2.1 k 0.2 3.1 f 0.2

of “C-AA TXB 0.4kO.1 2.4 f 0.2 4.4 + 0.3

PGD 0.9 f 0. I I .8 + 0.2 2.2 f 0.2

Percent of conversion of ‘%-AA to prostanoids are expressed by mean values + SEM. Five preparations tested in each group.

prostanoids in prepubertal animals (which was significantly lower than that of the other groups) was not further reduced by castration. It was reported that, at this age, testosterone serum levels are not affected by castration (6). Since these findings have suggested a relationship between androgens and prostanoid production by the vas deferens, we studied the effects of substitution therapy with testosterone propionate on castrated animals. This treatment produced a significant increase in prostanoid synthesis by pubertal and adult castrated rats, reaching levels similar to those observed in controls of the same age. Indeed, comparable results were obtained with the same androgen therapy on normal prepubertal animals, which displayed prostanoid production levels similar to those of castrated prepubertal and adults. These data are showing a direct relationship between the presence of androgens and prostanoid synthesis in vas deferens. To clarify if these actions of testosterone are specific for this hormone or are due to an effect on membrane fluidity, shared with other sex steroids (13), we treated adult castrated animals with 17-p estradiol and with progesterone. These steroids increased the 14C-AA conversion to prostanoids in vas deferens, but the fact that

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Prepubertal

measuring techniques, castration timing, testosterone dose administered and tissue incubation conditions. In summary, our results indicate that the rat isolated vas deferens produces at least four different prostanoids from 14C-AA, and that activity is dependent on the presence of testosterone.

Percent of AA Conversion

Acknowledgement Supported

by Grant 663X from CONICET.

References

PGF

PGE

TX82

PGD

Prostanoids D Fig. 3

Control

Testosterone

Effects of testosterone propionate treatment on conversion (l-‘%I AA in the vas deferens from intact prepubertal rats. Significantly different (*: p
of

the reached levels were lower than those observed in testosterone-treated castrated, suggests that the effects of the androgen would be at least in part specific. In this report, no significant variations between the different AA metabolites measured were found at the different ages or with castration or hormone treatments. In a previous paper (4) we have observed that only PGF,, synthesis and release was increased by castration, although in that case the PGs measured were synthesized from an endogenous substrate, and in the present study we are showing PG production from an exogenous substrate. Differences between these conditions in human lung cells have been described (14). PGE, was found to be more sensitive to hormonal deprivation than PGF2, in rat (5) and mouse (1) vas deferens. These disagreements between our data and those studies could be explained, at least in part, by different experimental conditions related to prostanoid

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