ANNUAL RHYTHMS OF LUTEINIZING
HORMONE, FOLLICLE\x=req-\
STIMULATING HORMONE, PROLACTIN AND TESTOSTERONE IN THE SERUM OF MALE RHESUS MONKEYS W. BECK
AND
W. WUTTKE
Max-Planck-Institute for D-3400
Biophysical Chemistry, Department of Neurobiology, Göttingen-Nikolausberg, West Germany (Received 17 November 1978) SUMMARY
Six male rhesus monkeys were kept under rigidly controlled conditions for 1\p=n-\2years. During August of the first year a thyrotrophin releasing hormone (TRH) test was performed on each of the monkeys by giving 10 \g=m\g TRH as a bolus injection. Significantly increased serum prolactin levels occurred 15 min after the injection. After a training period of 2 months, during which blood samples were collected every other day by puncture of the saphenous vein, blood was collected three times a week for 14 months. Serum levels of prolactin, LH, FSH and testosterone were measured by radioimmunoassay. Mean serum prolactin levels increased significantly during June, July and August in all six animals. Peak levels were observed in August and September and then levels declined gradually to reach a minimum in April and May. Mean serum testosterone levels closely paralleled the annual pattern of prolactin. Mean serum LH levels significantly decreased during the time when mean serum prolactin and testosterone levels were increasing and they increased again at the time of decreasing mean prolactin levels, i.e. mean serum LH and prolactin were negatively correlated. In individual monkeys, however, a rigid negative correlation between serum prolactin and LH could not be demonstrated. Mean serum FSH levels did not change
significantly.
introduction
Pituitary prolactin release which is induced by stress has been shown to occur in a number of species (Ajika, Kalra, Fawcett, Krulich & McCann, 1972; Fenske & Wuttke, 1976).
Ferin, Carmel, Warren, Himsworth & Frantz (1976) measured the basal concentration of
prolactin
in restrained rhesus monkeys during a period of 5 h but little is known of the prolactin levels of the capturing of monkeys in their cages. The effects of such stressful manipulation on the plasma prolactin levels in male rhesus monkeys has been considered. A number of investigations report that sexual behaviour in the male rhesus monkey shows a marked annual rhythm (Plant, Zumpe, Sauls & Michael, 1974; Michael & Zumpe, 1976; Michael & Zumpe, 1978). Since sexual behaviour is androgen-dependent it has been assumed that serum levels of testosterone also undergo seasonal changes (Michael & Zumpe, 1978). An annual rhythm has also been observed in male rhesus monkeys kept under controlled laboratory conditions (Plant et al. 1974). However, it is not known whether serum levels of gonadotrophins and of prolactin also show a similar annual rhythm which might cause the rhythm in the concentrations of serum testosterone. Preliminary experiments in our laboratory have revealed that serum prolactin levels show a clear seasonal rhythm, suggesting that prolactin might be involved in the regulation of possible acute effect on
of luteinizing hormone (LH) or of gonadal steroids (Beck, Hancke & Wuttke, It 1978). has been shown that prolactin not only modifies pituitary LH release but that it also alters steroidogenesis in the testicular tissue in the rat (Bartke & Dalterio, 1976; Beck, Engelbart, Gelato & Wuttke, 1977; Beck & Wuttke, 1977) and in man (Bohnet, Dahlèn, Wuttke & Schneider, 1976). This study has, therefore, attempted to establish whether there are annual rhythms in the levels of serum prolactin, LH, follicle-stimulating hormone (FSH) and testosterone in the male rhesus monkey.
rhythms
materials and methods
Six adult male rhesus monkeys each weighing 5-8 kg were quarantined for 3-6 months under natural daylight conditions. Upon arrival in the laboratory, they were kept under controlled conditions for at least 1 year. They were housed in individual cages in an airconditioned room (24 ± 1 °C) with the lights on from 07.00 to 20.00 h. In August of the initial year of adaptation, each monkey was put into a primate chair for 4 h. Immediately after the monkeys had been installed in the chair, a venous catheter was fixed into the basilic vein and blood samples (2 ml) were collected every 10 min for 120 min. After this period of 120 min 10 µg synthetic thyrotrophin releasing hormone (TRH; Relefact; Hoechst, Frankfurt, West Germany) dissolved in 2 ml Ringer's solution were intravenously injected and blood (2 ml each time) was collected at intervals of 5 min for 20 min and then at intervals of 10 min for the subsequent 100 min. After a training period of 2 months, during which blood was collected every other day by puncture of the saphenous vein, the monkeys were so well trained that they did not show any signs of stress throughout the bleeding procedure. Blood samples (2-5 ml) were then collected from the saphenous vein three times a week (between 09.00 and 11.00 h) for 14 months (from February 1976 to April 1977). Each monkey was provided with food supple¬ mented with vitamins and minerals, and water ad libitum. Haemoglobin and erythrocytes were checked at monthly intervals. Serum prolactin was measured using a heterologous radioimmunoassay as described by Hwang, Guyda & Friesen (1971). This monkey prolactin assay has been validated by Josimovich, Weiss & Hutchinson (1974) and in preliminary studies performed before the present experiments we demonstrated parallelism between dilutions of the human reference preparation, human serum, extracts of monkey pituitary glands and monkey serum. Serum LH and FSH were measured as described and validated by Niswender, Monroe, Peckham, Midgley, Knobil & Reichert (1971) and by Boorman, Niswender, Gay, Reichert & Midgley (1973). The rat LH reference preparation and the human FSH preparation (LER 907) were supplied by NIAMDD, Bethesda, Maryland, U.S.A. The reference preparation for prolac¬ tin was human prolactin (75.7.28) provided by Dr H. Friesen (Winnipeg, Canada). Serum testosterone was measured by a radioimmunoassay as described by Döhler & Wuttke (1976). The hormone concentrations in all sera from each animal were assayed in one radio¬ immunoassay for a given hormone. The intra-assay variance for all hormones measured in the present study varied by no more than 8%. RESULTS
The effects of stress upon serum prolactin levels were investigated (Fig. 1). Measurements were made immediately after the removal of the animal from its cage and before and after the injection of TRH. Serum prolactin levels were raised at the beginning of the test period and they declined to basal values within 60-90 min ( < 0 5). The levels increased signifi¬ cantly (P< 0-005) within 5 min of the injection of TRH. Changes in mean serum prolactin levels during the 14 month investigation period are shown in Fig. 2a. Very low prolactin levels were observed during April and May, but they rose progressively from June to September to reach peak levels in October. These peak
60
50
S 40 ö
3020 10-
— -1-1-1-1-1-1-1-1-1-1-r
~
~80~
— — —
0 120 -40 -20 20 40 100 Time before and after injection (min) Fig. 1. Serum prolactin levels before and after the injection of 10 µg thyrotrophin releasing hormone (TRH; shown by arrow). Six rhesus monkeys were placed in primate chairs and the saphenous vein was punctured at —120 min. Note the decreasing prolactin values during the first 60 min. Values are means ± s.e.m.
-120 -100 -80
-60
levels were 20-fold higher than the levels observed in April and May. From October to March prolactin levels declined significantly but the minimum was not quite as low as that measured in the preceding year. Mean serum LH levels (Fig. 2b) were high at the beginning of the test period and lowest values were observed between July and October. From November to February mean LH values increased again (P
HORMONE, FOLLICLE\x=req-\
STIMULATING HORMONE, PROLACTIN AND TESTOSTERONE IN THE SERUM OF MALE RHESUS MONKEYS W. BECK
AND
W. WUTTKE
Max-Planck-Institute for D-3400
Biophysical Chemistry, Department of Neurobiology, Göttingen-Nikolausberg, West Germany (Received 17 November 1978) SUMMARY
Six male rhesus monkeys were kept under rigidly controlled conditions for 1\p=n-\2years. During August of the first year a thyrotrophin releasing hormone (TRH) test was performed on each of the monkeys by giving 10 \g=m\g TRH as a bolus injection. Significantly increased serum prolactin levels occurred 15 min after the injection. After a training period of 2 months, during which blood samples were collected every other day by puncture of the saphenous vein, blood was collected three times a week for 14 months. Serum levels of prolactin, LH, FSH and testosterone were measured by radioimmunoassay. Mean serum prolactin levels increased significantly during June, July and August in all six animals. Peak levels were observed in August and September and then levels declined gradually to reach a minimum in April and May. Mean serum testosterone levels closely paralleled the annual pattern of prolactin. Mean serum LH levels significantly decreased during the time when mean serum prolactin and testosterone levels were increasing and they increased again at the time of decreasing mean prolactin levels, i.e. mean serum LH and prolactin were negatively correlated. In individual monkeys, however, a rigid negative correlation between serum prolactin and LH could not be demonstrated. Mean serum FSH levels did not change
significantly.
introduction
Pituitary prolactin release which is induced by stress has been shown to occur in a number of species (Ajika, Kalra, Fawcett, Krulich & McCann, 1972; Fenske & Wuttke, 1976).
Ferin, Carmel, Warren, Himsworth & Frantz (1976) measured the basal concentration of
prolactin
in restrained rhesus monkeys during a period of 5 h but little is known of the prolactin levels of the capturing of monkeys in their cages. The effects of such stressful manipulation on the plasma prolactin levels in male rhesus monkeys has been considered. A number of investigations report that sexual behaviour in the male rhesus monkey shows a marked annual rhythm (Plant, Zumpe, Sauls & Michael, 1974; Michael & Zumpe, 1976; Michael & Zumpe, 1978). Since sexual behaviour is androgen-dependent it has been assumed that serum levels of testosterone also undergo seasonal changes (Michael & Zumpe, 1978). An annual rhythm has also been observed in male rhesus monkeys kept under controlled laboratory conditions (Plant et al. 1974). However, it is not known whether serum levels of gonadotrophins and of prolactin also show a similar annual rhythm which might cause the rhythm in the concentrations of serum testosterone. Preliminary experiments in our laboratory have revealed that serum prolactin levels show a clear seasonal rhythm, suggesting that prolactin might be involved in the regulation of possible acute effect on
of luteinizing hormone (LH) or of gonadal steroids (Beck, Hancke & Wuttke, It 1978). has been shown that prolactin not only modifies pituitary LH release but that it also alters steroidogenesis in the testicular tissue in the rat (Bartke & Dalterio, 1976; Beck, Engelbart, Gelato & Wuttke, 1977; Beck & Wuttke, 1977) and in man (Bohnet, Dahlèn, Wuttke & Schneider, 1976). This study has, therefore, attempted to establish whether there are annual rhythms in the levels of serum prolactin, LH, follicle-stimulating hormone (FSH) and testosterone in the male rhesus monkey.
rhythms
materials and methods
Six adult male rhesus monkeys each weighing 5-8 kg were quarantined for 3-6 months under natural daylight conditions. Upon arrival in the laboratory, they were kept under controlled conditions for at least 1 year. They were housed in individual cages in an airconditioned room (24 ± 1 °C) with the lights on from 07.00 to 20.00 h. In August of the initial year of adaptation, each monkey was put into a primate chair for 4 h. Immediately after the monkeys had been installed in the chair, a venous catheter was fixed into the basilic vein and blood samples (2 ml) were collected every 10 min for 120 min. After this period of 120 min 10 µg synthetic thyrotrophin releasing hormone (TRH; Relefact; Hoechst, Frankfurt, West Germany) dissolved in 2 ml Ringer's solution were intravenously injected and blood (2 ml each time) was collected at intervals of 5 min for 20 min and then at intervals of 10 min for the subsequent 100 min. After a training period of 2 months, during which blood was collected every other day by puncture of the saphenous vein, the monkeys were so well trained that they did not show any signs of stress throughout the bleeding procedure. Blood samples (2-5 ml) were then collected from the saphenous vein three times a week (between 09.00 and 11.00 h) for 14 months (from February 1976 to April 1977). Each monkey was provided with food supple¬ mented with vitamins and minerals, and water ad libitum. Haemoglobin and erythrocytes were checked at monthly intervals. Serum prolactin was measured using a heterologous radioimmunoassay as described by Hwang, Guyda & Friesen (1971). This monkey prolactin assay has been validated by Josimovich, Weiss & Hutchinson (1974) and in preliminary studies performed before the present experiments we demonstrated parallelism between dilutions of the human reference preparation, human serum, extracts of monkey pituitary glands and monkey serum. Serum LH and FSH were measured as described and validated by Niswender, Monroe, Peckham, Midgley, Knobil & Reichert (1971) and by Boorman, Niswender, Gay, Reichert & Midgley (1973). The rat LH reference preparation and the human FSH preparation (LER 907) were supplied by NIAMDD, Bethesda, Maryland, U.S.A. The reference preparation for prolac¬ tin was human prolactin (75.7.28) provided by Dr H. Friesen (Winnipeg, Canada). Serum testosterone was measured by a radioimmunoassay as described by Döhler & Wuttke (1976). The hormone concentrations in all sera from each animal were assayed in one radio¬ immunoassay for a given hormone. The intra-assay variance for all hormones measured in the present study varied by no more than 8%. RESULTS
The effects of stress upon serum prolactin levels were investigated (Fig. 1). Measurements were made immediately after the removal of the animal from its cage and before and after the injection of TRH. Serum prolactin levels were raised at the beginning of the test period and they declined to basal values within 60-90 min ( < 0 5). The levels increased signifi¬ cantly (P< 0-005) within 5 min of the injection of TRH. Changes in mean serum prolactin levels during the 14 month investigation period are shown in Fig. 2a. Very low prolactin levels were observed during April and May, but they rose progressively from June to September to reach peak levels in October. These peak
60
50
S 40 ö
3020 10-
— -1-1-1-1-1-1-1-1-1-1-r
~
~80~
— — —
0 120 -40 -20 20 40 100 Time before and after injection (min) Fig. 1. Serum prolactin levels before and after the injection of 10 µg thyrotrophin releasing hormone (TRH; shown by arrow). Six rhesus monkeys were placed in primate chairs and the saphenous vein was punctured at —120 min. Note the decreasing prolactin values during the first 60 min. Values are means ± s.e.m.
-120 -100 -80
-60
levels were 20-fold higher than the levels observed in April and May. From October to March prolactin levels declined significantly but the minimum was not quite as low as that measured in the preceding year. Mean serum LH levels (Fig. 2b) were high at the beginning of the test period and lowest values were observed between July and October. From November to February mean LH values increased again (P
