Depression of nocturnal pineal serotonin N-acetyltransferase activity in castrate male rats

JoumaZof J. Neural Transmission 48, 1--8 (1980) 9 by Springer-Verlag 1980

Depression of Nocturnal Pineal Serotonin N-acetyltransferase Activity in Castrate Male Rats P. K. Rudeen and R. J. Reiter Department of Anatomy, Bowman Gray School of Medicine, Winston-Salem, North Carolina, and Department of Anatomy, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, U.S.A. With 1 Figure Received January 7, 1980

Summary Pineal serotonin N-acetyltransferase (NAT) activity was examined in intact rats, castrated rats, and in rats that had been castrated and had received testosterone proprionate. Castration resulted in significantly depressing nocturnal levels of pineal NAT (p < 0.05) when compared to enzyme activity in intact rats. Testosterone proprionate administration restored plasma LH levels to normal values in castrate rats but did not induce nocturnal pineal enzyme activity to levels seen in the pineal glands of intact rats. The data substantiate the existence of a feedback control of pineal biosynthetic activity by the hypophyseal-gonadal system, but the identity of the hormone(s) responsible for regulation of pineal NAT activity is not known. Key words: Pineal NAT, castration, gonads.

Introduction A multitude of reports describes the effects that pineal gland compounds have upon various aspects of reproductive physiology (Reiter, 1978). There are also a number of reports suggesting an effect of gonadal and pituitary hormones on pineal gland physiology. In 1965, Vivien observed that gonadotrophins enhanced the synthetic activity of the pineal gland, and Karasek (1971) observed that hypophysectomy reduced the ultrastructural correlates of pineal gland hormone synthesis. Karasek et al. (1976) suggested that either t 5ournaiof Neural Transmission48/I 0300-9564/80/0048/0001/~ 01.60

p. K. Rudeen and R. J. Reiter: gonadotrophins or gonadal steroids result in feedba& control on the production of pineal hormones. More direct evidence that the biosynthesis of pineal hormones responds to changes in reproductive physiology is offered by investigations of the enzyme involved in melatonin synthesis. Melatonin, 5-methoxy-N-acetyltryptamine, has been considered to be a pineal hormone with antigonadal properties (Reiter, 1978). Pineal serotonin N-acetyltransferase (NAT: E.C. 2.3.1.5) N-acetylates serotonin to form N-acetylserotonin (Weissbach et al., 1960). Melatonin is formed by the O-methylation of N-acetylserotonin by the enzyme hydroxyindole-O-methyltransferase (HIOMT: E.C. 2.1.1.4) (Axelrod and Weissbach, 1961). The activity of N A T in the rat pineal gland is used as an index of melatonin formation since the activity of this enzyme is involved in the biosynthetic pathway in melatonin formation (Klein et al., 1970). A close correlation between nocturnal pineal N A T activity and the appearance of nocturnal pineal melatonin has been demonstrated in rats (Wilkinson et al., 1977). Rat pineal N A T activity exhibits a marked diurnal variation being 15--60 times greater during darkness than during light (Klein and Weller, 1970; Rudeen et al., 1975) and is dependent on norepinephrine stimulation of adenyl cyclase activity (Klein et aI., 1970). There are several reports on the effects of castration on pineal enzyme activity. Houssay and Barcelo (1972) examined the effects of castration on H I O M T activity. Neither castration nor testosterone administration significantly altered H I O M T activity. The authors concluded that the effects of castration on the pineal, as formerly described, were independent of melatonin formation. On the other hand, Nagle et al. (1974) observed that pineal H I O M T activity was inhibited a~er castration of male rats, and furthermore, the administration of testosterone in low doses restored pineal enzyme activity. These reports indicate that the activity of at least one pineal enzyme, HIOMT, may be altered by castration. However, data are lacking in which the activity of pineal N A T is affected by castration or by gonadal steroids. The purpose of this study is to investigate the effects of castration and testosterone administration on pineal N A T during its period of greatest activity. Effects of castration on the activity of this enzyme would substantiate the influence of the reproductive organ hormones on melatonin formation. Methods

Adult male Sprague-Dawiey rats (125--150 gram) from the Holtzman Company (Madison, Wisconsin) were used in this experiment. The animals

Pineal NAT in Castrate Rats

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were allowed to acclimate to a 14-hour photoperiod (light 9 d a r k - L : D 14:10) for two weeks prior to castration. The animals quarters were maintained at 23 + 2 ~ having food (Wayne Lab Blox) and tap water available ad libitum. After acclimation the animals were randomly selected into 5 groups according to their treatment as follows: (1) (2) (3) (4) (5)

Intact Castrate Intact + Sham-implant Castrate + Sham-implant Castrate + Testosterone-implant

The rats were maintained in the conditions described for 25 days after castration. A testosterone proprionate pellet (50 mg) was implanted subcutaneously into appropriate animals at the time of castration. A pellet made from an equivalent amount of bovine serum albumin powder was implanted subcutaneously into animals denoted as having sham-implants. At the time of autopsy, the animals were killed by decapitation at 4 a.m. during the dark phase. Blood was collected into individual heparinized tubes and plasma was harvested and stored frozen ( - 2 0 ~ until analysis for luteinizing hormone (LH) concentration. Pineal glands were rapidly dissected from the skull, placed into individual foils, frozen on solid CO2 and stored at - 2 0 ~ until assay. The entire procedure was completed by the aid of a dim red light (Kodak Safelamp, 15 watt; 1A red filter). Pineal serotonin NAT activity was estimated by a modification of the method described by Deguchi and Axelrod (1972). Modifications of this assay are described elsewhere (Rudeen and Reiter, 1977). Pineal NAT activity is expressed as nmoles of 14C-N-acetyltryptamine synthesized per pineal gland per hour. Plasma LH was measured-using a modified radioimmunoassay procedure described by Goldman and Porter (1970). Reagents for this assay was provided by the Rat Hormone Distribution Program, NIAMDD. LH values are reported in terms of NIAMDD Rat LH RP-1. The data were analyzed by an analysis of variance (ANOVA), and by a t-test for differences among several means (Bruning and Kintz, 1977).

Results Nocturnal pineal N A T activity in castrate male rats was significantly lower than that of intact rats (Fig. 1 A; p < 0.05). The nocturnal enzyme activity was lower but not significantly reduced in rats that had been castrated and received the sham-pellet. Interestingly, castrate animals having received testosterone proprionate exhibited an equivalent depression of nocturnal pineal N A T activity as the castrate animals that received the sham-pellet. Though the reduction of enzyme activity after castration was one half the values observed in intact animals, variability within the groups prevented 1*

P. K. Rudeen and R. J. Reiter:

the statistical observation of a significant difference among intact animals and castrate animals receiving implants. Plasma LH levels were significantly elevated in castrate animals (p < 0.001) when compared to intact animals (Fig. 1 B). Furthermore, plasma LH was also significantly enhanced in animals that were castrate but received the sham-pellet ( p < 0 . 0 0 1 ) . Testosterone proprionate administration to castrate rats reduced plasma LH levels to normal values. The pineal N A T activity and plasma LH levels in intact rats implanted with sham-pellets were similar to the values observed in intact rats that did not receive the implants. vs intact

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