EFFECT OF LUTEINIZING HORMONE RELEASING HORMONE ON PLASMA LEVELS OF LUTEINIZING HORMONE, OESTRADIOL AND TESTOSTERONE IN THE MALE DOG
GWYNETH E. JONES, KAY BAKER, DIAN R. FAHMY AND A. R. BOYNS Teño vus Institute for Cancer Research, Welsh National School of Medicine, The Heath, Cardiff, CF4 4XX
(Received 25 July 1975) SUMMARY
An injection of luteinizing hormone releasing hormone (LH-RH) increased plasma LH and testosterone concentrations in the male dog, but no significant increase in plasma oestradiol\x=req-\ 17\g=b\ levels was observed. Repeated injections of LH-RH produced an increase in plasma LH levels but there was a progressive decline in the response with each injection. The concentration of plasma testosterone reached a maximum within 40 min of the first injection of LH-RH and remained constant thereafter while plasma oestradiol concentration gradually increased with successive injections of LH-RH.
INTRODUCTION
It is
generally believed that testosterone plays a major role in the regulation of luteinizing hormone (LH) secretion in the male animal. Thus the removal of the testes results in a rapid increase in plasma LH and follicle-stimulating hormone (FSH) levels (Ramirez & McCann, 1965; Gay & Midgley, 1969) while the administration of testosterone reverses this effect (Verjans, Eik-Nes, Aafjes, Veis & van der Molen, 1974). However oestradiol-17/? benzoate is more potent than testosterone propionate in suppressing serum gonadotrophin levels (Verjans et al. 1974). Furthermore, since testosterone does not serve as a complete physio¬ logical replacement after removal of the testes (Ramirez & McCann, 1965; Steinberger & Duckett, 1968; Gay & Bogdanove, 1969), it has been suggested that oestrogen may play an important role in the reproductive function of male animals (Gans & Van Rees, 1962). In relation to this, it has been reported that oestradiol-17/? inhibits luteinizing hormone re¬ leasing hormone (LH-RH)-induced LH release in men (Cole, Llewelyn, Link & Boyns, 1974) and in male dogs (Jones & Boyns, 1974). The inhibitory effect can be observed with i.v. doses of oestradiol-17/? as low as 100 ng and within 1 h of the injection. Oestradiol17/? also significantly reduced the basal plasma LH concentration before the LH-RH in¬ jection (Jones & Boyns, 1976); androgens were without effect (Jones & Boyns, 1974). It would seem reasonable to assume therefore that if a regulating feedback control system exists it should be expected that the administration of LH-RH would promote secretion of testosterone and oestradiol-17/?. This report describes the investigation of changes in the concentration of oestradiol after LH-RH administration to the male dog.
MATERIALS AND METHODS
Measurement of plasma hormones Canine plasma LH was determined by a double antibody radioimmunoassay (Boyns, Jones, Bell, Christie & Parkes, 1972) using purified canine LH (SEPH-2) as standard. Plasma oestradiol-17/? was measured by a modification of the radioimmunoassay described by Cameron & Jones (1972), and testosterone by the radioimmunoassay method of Hillier, Brownsey & Cameron (1973). Sample collection Dr H. Synthetic LH-RH, prepared by Gregory (ICI Ltd, Cheshire) according to the deca¬ Baba, Nair, Arimura & Schally (1971), was dis¬ sequence Matsuo, peptide proposed by solved in 0-15 M-NaCl for injection. Themalon (3-dimethyl-amino-l,l-dithien-2-yl-but-l-ene hydrochloride ; Burroughs Wellcome Ltd) was dissolved in water for injection. Seven intact male beagle dogs (aged between 4 and 6 years) were fed with pellets (Dugdale's Wuffitmix) and water. Dogs were sedated by administration (s.c.) of 250 mg Themalon irrespective of body wt, and 30 min later an indwelling cannula (Venflon 17-gauge) was introduced into a superficial leg vein and taped in position. A three-way stopcock was attached and the cannula was filled with heparin solution in 0-15M-NaCl (30 i.u./ml). Blood samples (5 ml) were obtained after first aspirating the heparin solution from the catheter. After removal of the blood the catheter was refilled with heparin. The blood was mixed with 1 mg EDTA/ml and retained at 4 °C. At the end of the experiment the samples were centrifuged; plasma was collected and stored at —20 °C until assayed. To study the effect of a single LH-RH injection, blood samples were collected at —10, 0, 60 and 120 min intervals after introduction of the cannula. After 165 min, LH-RH (5 µg) was injected through the cannula and samples were then taken at 5 min intervals for 25 min then every 15 min until the end of the experiment. Further studies were carried out 1 month later to investigate the effect of repeated injections of LH-RH. Blood samples were taken at —10, —5 and 0 min, continuing at 5 min intervals up to 110 min. LH-RH (5/ig) was administered at 0, 30, 60 and 90 min after the commencement of the experiment. The mean ( +s.e.m.) plasma LH, oestradiol and testosterone concentrations for each time interval were calculated. RESULTS
Effect of single injection of LH-RH Plasma LH concentration increased within 5 min of the injection of LH-RH, reaching a maximum after 15 min. There was an insignificant increase in the concentration of plasma oestradiol-17/?. However, plasma testosterone increased 20 min after injection of LH-RH, the maximum level being reached after 40 min. Thereafter, the plasma testosterone con¬ centration slowly decreased (Fig. 1). a
Effect of repeated injections of LH-RH each Although injection of LH-RH produced an increase in plasma LH concentration, there was a trend towards a diminished response to LH-RH with each subsequent injection. The plasma oestradiol-17/? concentration however, showed a gradual increase with suc¬ cessive injections of LH-RH. The plasma testosterone concentration increased after the first injection of LH-RH (Fig. 2). The increase was sustained at a concentration of approxi¬ mately 3-8 ng/ml from 40 min after the first injection to the end of the experiment.
7 6 5 4
3
H
2 1
40 o
5
301
120
60
| 165
| 190 205 235 250 220
S "S
~ -160
— —|— — — — 235 250 120 165 190 205 220 Time after cannulation (min)
Fig. 1. Effect of a single injection of 5 /ig luteinizing hormone releasing hormone (LH-RH) (arrows) on plasma concentrations of LH, oestradiol-17/?, and testosterone in dogs. Values are means ± s.e.m. ; «
=
6.
DISCUSSION
Longcope (1973) has shown that plasma concentrations of oestradiol- ß, oestrone, testosterone and androstenedione increase in both young and old men following two daily injections of human chorionic gonadotrophin (HCG). Weinstein, Kelch, Jenner, Kaplan efe Grumbach (1974) also reported that prolonged administration of HCG to men results in an eightfold increase in the plasma concentration of oestradiol-17/?, whereas testosterone increases only fivefold. Furthermore, Fishman, Sarfaty, Wilson & Lipsett (1967) demon¬ strated that the relative increase of the oestradiol production rate always exceeded that of testosterone after administration of HCG. In contrast however, Kelch, Jenner, Weinstein, Kaplan & Grumbach (1972) reported that although canine testes secreted oestradiol, pro¬ longed administration of HCG to dogs did not result in a rise in oestradiol concentrations in peripheral or spermatic venous blood.
10 20 30 40 50 60 70 80 90 100 110
10 20 30 40 50 60 70 80 90 100110
Time (min) Fig. 2. Effect of repeated injections of 5 //g luteinizing hormone releasing hormone (LH-RH) (arrows) on plasma concentrations of LH, oestradiol-17/?, and testosterone in dogs. Values are means± s.e.m.;
=
1.
In the work reported here, plasma LH levels increased within 5 min of a single injection of LH-RH. The plasma testosterone concentration had risen by 20 min and reached a maxi¬ mum within 40 min of LH-RH administration. This increase would appear faster than that observed in man. Roth, Grumbach & Kaplan (1973) found that the plasma testosterone concentration reached a maximum 4 h after LH-RH administration to pubertal and adult men and Judd, Rebar, Vandenberg & Yen (1974) reported a significant increase in plasma testosterone and androstenedione in men 30-60 min after LH-RH administration with maximum levels being reached after 120 min. The increase in plasma testosterone concentration must be due to the effect of LH-RH on the pituitary via LH/FSH secretion since there appears to be no direct effect of LH-RH on Leydig cell function (Suzuki, Takahashi, Lin & Asarro, 1970). The single injection of 5 µg LH-RH to the dogs had a negligible effect on the oestradiol-17/? concentration of the plasma. However, Wagner, Böckel, Hrubesch & Grote (1972) reported that a single injection of 25 µg LH-RH doubled serum oestradiol-17/? concentrations within 2 h in men. Similarly, Kastin, Schally, Schalch, Korenman, Miller, Guai & Perez-Pasten (1972) found that plasma oestradiol levels increase to four times those of basal levels 32 min after giving LH-RH to men.
Repeated injections of LH-RH into dogs at 30 min intervals produced an increase in plasma testosterone levels which reached a maximum within 40 min after the first injection. It appears that maximum secretion of testosterone is reached fairly rapidly after LH-RH administration. Katongole, Naftolin & Short (1971) also found that in the bull, sexual stimulation would increase plasma testosterone levels when plasma testosterone was low, but would not do so if testosterone levels were already high. This increase in testosterone may be due to maximal stimulation of the testes or a new equilibrium being reached in testosterone secretion due to the negative feedback effect of LH. Repeated injections of LH-RH produced an increase in plasma LH levels but a gradual decline in the response after each injection. This is in contrast with experiments in men where three i.v. injections of LH-RH at 60 min intervals have been reported to produce a progressive increase in LH response after each injection (Wagner et al. 1972). The decline in the LH response may indicate a loss of pituitary responsiveness to LH-RH, or a reduction of the pituitary reserves of LH. It could also be due, however, to the gradual increase of plasma oestradiol concentrations observed with successive injections of LH-RH. Although complete inhibition of the LH response to LH-RH was not observed when oestradiol levels were maximal, plasma LH levels were at a minimum at this point. Since a latent period, of about 60 min, is needed for oestradiol-17/? to inhibit the LH response to LH-RH (Jones & Boyns, 1976), the data suggest that this increasing plasma oestradiol concentration may be regulating LH secretion in the male dog. However, although androgens did not modify the pituitary response to LH-RH injection (Jones & Boyns, 1974), it still seems reasonable to assume that they can influence the mechanism at the hypothalamic level and inhibit LH-RH release.
grateful to the Tenovus Organisation for generous financial assistance, to Dr H. ICI Ltd, for the gift of synthetic LH-RH and to Dr A. Stockell Hartree for Gregory, canine LH. We thank Dr A. T. B. Edney (Pedigree Petfoods Ltd) for his help in purified this project; Mr B. Joyce for technical assistance and Mr D. Mulcuck for expert technical advice. We are grateful to Professor K. Griffiths for his support and encouragement. We
are
REFERENCES
Boyns, A. R., Jones, G. E., Beli, E. T., Christie, D. W. & Parkes, M. F. (1972). Development of a radio¬ immunoassay for canine luteinizing hormone. Journal of Endocrinology 55, 279-291. Cameron, E. H. D. & Jones, D. A. (1972). Some observations on the measurement of estradiol-17/? in human plasma by radioimmunoassay. Steroids 20, 737-759. Cole, E. N., Llewelyn, D. E. H., Link, J. & Boyns, A. R. (1974). Acute modulation of pituitary gonado¬ trophin secretion by oestradiol or testosterone in healthy men. Journal of Endocrinology 63, 251-252. Fishman, L. M., Sarfaty, G. ., Wilson, H. & Lipsett, M. B. (1967). The role of the testis in oestrogen production. Ciba Foundation Colloquia on Endocrinology 16, 156-170. Gans, E. & Van Rees, G. P. (1962). Effect of small doses of oestradiol benzoate on pituitary production and release of I.C.S.H. in gonadectomized male and female rats. Acta Endocrinologica 39, 245-252. Gay, V. L. & Bogdanove, E. M. (1969). Plasma and pituitary LH and FSH in the castrated rat following short-term steroid treatment. Endocrinology 84, 1132-1142. Gay, V. L. & Midgley, A. R., Jr. (1969). Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioimmunoassay. Endocrinology 84, 1359-1364. Hillier, S. G., Brownsey, B. G. & Cameron, E. H. D. (1973). Some observations on the determination of
testosterone in human plasma by radioimmunoassay using antisera raised against testosterone-3-BSA and testosterone-lla-BSA. Steroids 21, 735-754. Jones, G. E. & Boyns, A. R. (1974). Effect of gonadal steroids on the pituitary responsiveness to synthetic luteinizing hormone releasing hormone in the male dog. Journal of Endocrinology 61, 123-131. Jones, G. E. & Boyns, A. R. (1976). Inhibition by oestradiol of the pituitary response to luteinizing hormone releasing hormone in the dog. Journal of Endocrinology 68, 475-479. Judd, H. L., Rebar, R., Vandenberg, G. & Yen, S. S. C. (1974). Effect of luteinizing hormone-releasing factor on Leydig cell function. Journal of Clinical Endocrinology and Metabolism 38, 8-13. Kastin, A. J., Schally, A. V., Schalch, D. S., Korenman, S. G., Miller, M. C, Guai, C. & Perez-Pasten, E. (1972). Characterization of the hormonal responses to luteinizing hormone-releasing hormone (LH-RH) in prepubertal and adult subjects. Pediatrics Research 6, 481-486. Katongole, C. B., Naftolin, F. & Short, R. V. (1971). Relationship between blood levels of luteinizing hormone and testosterone in bulls, and the effects of sexual stimulation. Journal of Endocrinology 50, 457-466. Kelch, R. P., Jenner, M. R., Weinstein, R., Kaplan, S. L. & Grumbach, M. M. (1972). Estradiol and testo¬ sterone secretion by human, simian and canine testes in males with hypogonadism and in male pseudohermaphrodites with the feminizing testes syndrome. Journal of Clinical Investigation 51, 824-830. Longcope, C. (1973). The effect of human chorionic gonadotrophin on plasma steroid levels in young and old men. Steroids 21, 583-592. Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. & Schally, A. V. (1971). Structure of the porcine LHand FSH-releasing hormone. 1. The proposed amino acid sequence. Biochemical and Biophysical Research Communications 43, 1334-1337. Ramirez, V. D. & McCann, S. M. (1965). Inhibitory effect of testosterone on luteinizing hormone secretion in immature and adult rats. Endocrinology 76, 412-417. Roth, J. C, Grumbach, M. M. & Kaplan, S. L. (1973). Effect of synthetic luteinizing hormone-releasing factor on serum testosterone and gonadotropins in prepubertal, pubertal and adult males. Journal of Clinical Endocrinology and Metabolism 37, 680-686. Steinberger, E. & Duckett, G. (1968). Effect of testosterone propionate on the release of FSH from the pituitary gland. Acta Endocrinologica 57, 289-295. Suzuki, Y., Takahashi, M., Lin, Y. C. & Asarro, T. (1970). Rat intratesticular test systems for investigating luteinizing hormone releasing factor (LRF) and luteinizing hormone (LH) activities: effects of rat stalkmedian eminence extract and internal environment of rat testes upon rat and quail anterior pituitaries. Endocrinologica Japónica 17, 431-440. Verjans, H. L., Eik-Nes, K. B., Aafjes, J. H., Veis, F. J. M. & van der Molen, H. J. (1974). Effect of testo¬ sterone propionate, 5a-dihydrotestosterone propionate and oestradiol benzoate on serum levels of LH and FSH in the castrated adult male rat. Acta Endocrinologica 77, 643-654. Wagner, H., Böckel, ., Hrubesch, M. & Grote, G. (1972). Examination of the pituitary-gonadal relation¬ ship in man with synthetic LH/FSH-releasing hormone. In Hypothalamic hypophysiotropic hormones, pp. 257-270. Eds C. Guai & E. Rosenberg. Amsterdam: Excerpta Medica Foundation. Weinstein, R. L., Kelch, R. P., Jenner, M. R., Kaplan, S. L. & Grumbach, M. M. (1974). Secretion of unconjugated androgens and estrogens by the normal and abnormal human testis before and after human chorionic gonadotropa. Journal of Clinical Investigation 53, 1-6.
GWYNETH E. JONES, KAY BAKER, DIAN R. FAHMY AND A. R. BOYNS Teño vus Institute for Cancer Research, Welsh National School of Medicine, The Heath, Cardiff, CF4 4XX
(Received 25 July 1975) SUMMARY
An injection of luteinizing hormone releasing hormone (LH-RH) increased plasma LH and testosterone concentrations in the male dog, but no significant increase in plasma oestradiol\x=req-\ 17\g=b\ levels was observed. Repeated injections of LH-RH produced an increase in plasma LH levels but there was a progressive decline in the response with each injection. The concentration of plasma testosterone reached a maximum within 40 min of the first injection of LH-RH and remained constant thereafter while plasma oestradiol concentration gradually increased with successive injections of LH-RH.
INTRODUCTION
It is
generally believed that testosterone plays a major role in the regulation of luteinizing hormone (LH) secretion in the male animal. Thus the removal of the testes results in a rapid increase in plasma LH and follicle-stimulating hormone (FSH) levels (Ramirez & McCann, 1965; Gay & Midgley, 1969) while the administration of testosterone reverses this effect (Verjans, Eik-Nes, Aafjes, Veis & van der Molen, 1974). However oestradiol-17/? benzoate is more potent than testosterone propionate in suppressing serum gonadotrophin levels (Verjans et al. 1974). Furthermore, since testosterone does not serve as a complete physio¬ logical replacement after removal of the testes (Ramirez & McCann, 1965; Steinberger & Duckett, 1968; Gay & Bogdanove, 1969), it has been suggested that oestrogen may play an important role in the reproductive function of male animals (Gans & Van Rees, 1962). In relation to this, it has been reported that oestradiol-17/? inhibits luteinizing hormone re¬ leasing hormone (LH-RH)-induced LH release in men (Cole, Llewelyn, Link & Boyns, 1974) and in male dogs (Jones & Boyns, 1974). The inhibitory effect can be observed with i.v. doses of oestradiol-17/? as low as 100 ng and within 1 h of the injection. Oestradiol17/? also significantly reduced the basal plasma LH concentration before the LH-RH in¬ jection (Jones & Boyns, 1976); androgens were without effect (Jones & Boyns, 1974). It would seem reasonable to assume therefore that if a regulating feedback control system exists it should be expected that the administration of LH-RH would promote secretion of testosterone and oestradiol-17/?. This report describes the investigation of changes in the concentration of oestradiol after LH-RH administration to the male dog.
MATERIALS AND METHODS
Measurement of plasma hormones Canine plasma LH was determined by a double antibody radioimmunoassay (Boyns, Jones, Bell, Christie & Parkes, 1972) using purified canine LH (SEPH-2) as standard. Plasma oestradiol-17/? was measured by a modification of the radioimmunoassay described by Cameron & Jones (1972), and testosterone by the radioimmunoassay method of Hillier, Brownsey & Cameron (1973). Sample collection Dr H. Synthetic LH-RH, prepared by Gregory (ICI Ltd, Cheshire) according to the deca¬ Baba, Nair, Arimura & Schally (1971), was dis¬ sequence Matsuo, peptide proposed by solved in 0-15 M-NaCl for injection. Themalon (3-dimethyl-amino-l,l-dithien-2-yl-but-l-ene hydrochloride ; Burroughs Wellcome Ltd) was dissolved in water for injection. Seven intact male beagle dogs (aged between 4 and 6 years) were fed with pellets (Dugdale's Wuffitmix) and water. Dogs were sedated by administration (s.c.) of 250 mg Themalon irrespective of body wt, and 30 min later an indwelling cannula (Venflon 17-gauge) was introduced into a superficial leg vein and taped in position. A three-way stopcock was attached and the cannula was filled with heparin solution in 0-15M-NaCl (30 i.u./ml). Blood samples (5 ml) were obtained after first aspirating the heparin solution from the catheter. After removal of the blood the catheter was refilled with heparin. The blood was mixed with 1 mg EDTA/ml and retained at 4 °C. At the end of the experiment the samples were centrifuged; plasma was collected and stored at —20 °C until assayed. To study the effect of a single LH-RH injection, blood samples were collected at —10, 0, 60 and 120 min intervals after introduction of the cannula. After 165 min, LH-RH (5 µg) was injected through the cannula and samples were then taken at 5 min intervals for 25 min then every 15 min until the end of the experiment. Further studies were carried out 1 month later to investigate the effect of repeated injections of LH-RH. Blood samples were taken at —10, —5 and 0 min, continuing at 5 min intervals up to 110 min. LH-RH (5/ig) was administered at 0, 30, 60 and 90 min after the commencement of the experiment. The mean ( +s.e.m.) plasma LH, oestradiol and testosterone concentrations for each time interval were calculated. RESULTS
Effect of single injection of LH-RH Plasma LH concentration increased within 5 min of the injection of LH-RH, reaching a maximum after 15 min. There was an insignificant increase in the concentration of plasma oestradiol-17/?. However, plasma testosterone increased 20 min after injection of LH-RH, the maximum level being reached after 40 min. Thereafter, the plasma testosterone con¬ centration slowly decreased (Fig. 1). a
Effect of repeated injections of LH-RH each Although injection of LH-RH produced an increase in plasma LH concentration, there was a trend towards a diminished response to LH-RH with each subsequent injection. The plasma oestradiol-17/? concentration however, showed a gradual increase with suc¬ cessive injections of LH-RH. The plasma testosterone concentration increased after the first injection of LH-RH (Fig. 2). The increase was sustained at a concentration of approxi¬ mately 3-8 ng/ml from 40 min after the first injection to the end of the experiment.
7 6 5 4
3
H
2 1
40 o
5
301
120
60
| 165
| 190 205 235 250 220
S "S
~ -160
— —|— — — — 235 250 120 165 190 205 220 Time after cannulation (min)
Fig. 1. Effect of a single injection of 5 /ig luteinizing hormone releasing hormone (LH-RH) (arrows) on plasma concentrations of LH, oestradiol-17/?, and testosterone in dogs. Values are means ± s.e.m. ; «
=
6.
DISCUSSION
Longcope (1973) has shown that plasma concentrations of oestradiol- ß, oestrone, testosterone and androstenedione increase in both young and old men following two daily injections of human chorionic gonadotrophin (HCG). Weinstein, Kelch, Jenner, Kaplan efe Grumbach (1974) also reported that prolonged administration of HCG to men results in an eightfold increase in the plasma concentration of oestradiol-17/?, whereas testosterone increases only fivefold. Furthermore, Fishman, Sarfaty, Wilson & Lipsett (1967) demon¬ strated that the relative increase of the oestradiol production rate always exceeded that of testosterone after administration of HCG. In contrast however, Kelch, Jenner, Weinstein, Kaplan & Grumbach (1972) reported that although canine testes secreted oestradiol, pro¬ longed administration of HCG to dogs did not result in a rise in oestradiol concentrations in peripheral or spermatic venous blood.
10 20 30 40 50 60 70 80 90 100 110
10 20 30 40 50 60 70 80 90 100110
Time (min) Fig. 2. Effect of repeated injections of 5 //g luteinizing hormone releasing hormone (LH-RH) (arrows) on plasma concentrations of LH, oestradiol-17/?, and testosterone in dogs. Values are means± s.e.m.;
=
1.
In the work reported here, plasma LH levels increased within 5 min of a single injection of LH-RH. The plasma testosterone concentration had risen by 20 min and reached a maxi¬ mum within 40 min of LH-RH administration. This increase would appear faster than that observed in man. Roth, Grumbach & Kaplan (1973) found that the plasma testosterone concentration reached a maximum 4 h after LH-RH administration to pubertal and adult men and Judd, Rebar, Vandenberg & Yen (1974) reported a significant increase in plasma testosterone and androstenedione in men 30-60 min after LH-RH administration with maximum levels being reached after 120 min. The increase in plasma testosterone concentration must be due to the effect of LH-RH on the pituitary via LH/FSH secretion since there appears to be no direct effect of LH-RH on Leydig cell function (Suzuki, Takahashi, Lin & Asarro, 1970). The single injection of 5 µg LH-RH to the dogs had a negligible effect on the oestradiol-17/? concentration of the plasma. However, Wagner, Böckel, Hrubesch & Grote (1972) reported that a single injection of 25 µg LH-RH doubled serum oestradiol-17/? concentrations within 2 h in men. Similarly, Kastin, Schally, Schalch, Korenman, Miller, Guai & Perez-Pasten (1972) found that plasma oestradiol levels increase to four times those of basal levels 32 min after giving LH-RH to men.
Repeated injections of LH-RH into dogs at 30 min intervals produced an increase in plasma testosterone levels which reached a maximum within 40 min after the first injection. It appears that maximum secretion of testosterone is reached fairly rapidly after LH-RH administration. Katongole, Naftolin & Short (1971) also found that in the bull, sexual stimulation would increase plasma testosterone levels when plasma testosterone was low, but would not do so if testosterone levels were already high. This increase in testosterone may be due to maximal stimulation of the testes or a new equilibrium being reached in testosterone secretion due to the negative feedback effect of LH. Repeated injections of LH-RH produced an increase in plasma LH levels but a gradual decline in the response after each injection. This is in contrast with experiments in men where three i.v. injections of LH-RH at 60 min intervals have been reported to produce a progressive increase in LH response after each injection (Wagner et al. 1972). The decline in the LH response may indicate a loss of pituitary responsiveness to LH-RH, or a reduction of the pituitary reserves of LH. It could also be due, however, to the gradual increase of plasma oestradiol concentrations observed with successive injections of LH-RH. Although complete inhibition of the LH response to LH-RH was not observed when oestradiol levels were maximal, plasma LH levels were at a minimum at this point. Since a latent period, of about 60 min, is needed for oestradiol-17/? to inhibit the LH response to LH-RH (Jones & Boyns, 1976), the data suggest that this increasing plasma oestradiol concentration may be regulating LH secretion in the male dog. However, although androgens did not modify the pituitary response to LH-RH injection (Jones & Boyns, 1974), it still seems reasonable to assume that they can influence the mechanism at the hypothalamic level and inhibit LH-RH release.
grateful to the Tenovus Organisation for generous financial assistance, to Dr H. ICI Ltd, for the gift of synthetic LH-RH and to Dr A. Stockell Hartree for Gregory, canine LH. We thank Dr A. T. B. Edney (Pedigree Petfoods Ltd) for his help in purified this project; Mr B. Joyce for technical assistance and Mr D. Mulcuck for expert technical advice. We are grateful to Professor K. Griffiths for his support and encouragement. We
are
REFERENCES
Boyns, A. R., Jones, G. E., Beli, E. T., Christie, D. W. & Parkes, M. F. (1972). Development of a radio¬ immunoassay for canine luteinizing hormone. Journal of Endocrinology 55, 279-291. Cameron, E. H. D. & Jones, D. A. (1972). Some observations on the measurement of estradiol-17/? in human plasma by radioimmunoassay. Steroids 20, 737-759. Cole, E. N., Llewelyn, D. E. H., Link, J. & Boyns, A. R. (1974). Acute modulation of pituitary gonado¬ trophin secretion by oestradiol or testosterone in healthy men. Journal of Endocrinology 63, 251-252. Fishman, L. M., Sarfaty, G. ., Wilson, H. & Lipsett, M. B. (1967). The role of the testis in oestrogen production. Ciba Foundation Colloquia on Endocrinology 16, 156-170. Gans, E. & Van Rees, G. P. (1962). Effect of small doses of oestradiol benzoate on pituitary production and release of I.C.S.H. in gonadectomized male and female rats. Acta Endocrinologica 39, 245-252. Gay, V. L. & Bogdanove, E. M. (1969). Plasma and pituitary LH and FSH in the castrated rat following short-term steroid treatment. Endocrinology 84, 1132-1142. Gay, V. L. & Midgley, A. R., Jr. (1969). Response of the adult rat to orchidectomy and ovariectomy as determined by LH radioimmunoassay. Endocrinology 84, 1359-1364. Hillier, S. G., Brownsey, B. G. & Cameron, E. H. D. (1973). Some observations on the determination of
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