Department of Clinical Chemistry Max-Planck-lnstitut für Psychiatric, 8 München 40, Kraepelinstr. ¡0 Dermatologische Klinik der Technischen Universität, München FRG
FURTHER STUDIES ON HYPOTHALAMIC-PITUITARY-TESTICULAR FUNCTION IN OLD RATS
By Karl M. Pirke, Beate
Krings
and
Hermann-J. Vogt
ABSTRACT
dysfunction of the hypothalamic-pituitary-gonadal axis in old age studied in 24-month old male Wistar rats which were compared with 3-month old animals. The hypothalamic LH-RH content and the pituitary LH were significantly lower in the old than in the young adult animals. The plasma concentrations of LH and testosterone were significantly higher in the young rats. The primary cause of these age-dependent changes probably is a hypothalamic dysfunction. When isolated Leydig cells of young and old rats were incubated in vitro, the testosterone secretion per cell was significantly smaller in old than in young cells with as well as without HCG stimulation. In vivo stimulation of rats by iv injection of biologically active iodinated hCG revealed that the intratesticular uptake of the gonadotrophin was not different in young and old rats. The testosterone response, however, was significantly reduced in old age. An in vitro "desensitisation" experiment in which the LH receptor capacity was artificially reduced demonstrated that the 40 % reduction of receptor capacity in old testes as described earlier will not impair the testicular uptake of gonadotrophin from blood. Repeated injection of hCG results in equally elevated testosterone concentrations in young and old rats. The was
A decrease in
has been observed in the aging male rat Chan al. et 1977; Pirke et al. 1978 ). Although an age (Ghanadian et al. 1975; dependent fall in plasma LH indicates that the decline of the Leydig cell function is mainly caused by a pituitary or hypothalamic malfunction (Shaar et al 1975; Riegle 8c Mettes 1976; Pirke et al. 19786), other mechanisms may also be involved in reducing the testosterone production. We have recently
plasma
testosterone
described (Pirke et al. 1978 ) that the number of LH receptors as determined in vitro is significantly decreased in the testes of old rats. As a consequence, the testicular binding of LH and its effect on the Leydig cells may be altered in old animals. We have therefore studied the testicular response to stimulation by gonadotrophins in greater detail. We have examined the effect of hCG on isolated Leydig cells of young and old rats in vitro and the in vivo response to a single iv injection of HCG. We used iodinated hCG, which enabled us to follow the uptake and the binding of the gonadotrophic hormone. We have further studied the influence of repeated hCG injection into young adult and old rats.
MATERIALS AND METHODS
Animals Three-month old and 24-month old male Wistar rats were obtained from the Central Institute for Laboratory Animals, Hannover, Germany. The weight of the young rats ranged from 285 to 350 g (average: 3¡7 g). The average weight of the old rats was 435 g (range: 350 to 550 g). After the experiments all rats were studied by autopsy. All animals with atrophy or carcinoma of the testes or with other signs of severe illness were excluded from the studies. Four to 6 rats were housed in a cage under an 8-h dark, ¡6-h light schedule. Altrumine rat food and water were available ad libitum. Unless otherwise stated, the rats were sacrificed between 8:00 and ¡0:00 by decapita¬ tion immediately after removal from their cages. Trunk blood was collected. The hypothalamus and the pituitary gland were removed and snap frozen in acetone dry ice. Plasma and organs were stored at 30°C until analysed. -
Materials All reagents
were of analytical grade and obtained from Merck, Darmstadt unless otherwise stated. Collagenase (Typ III, 720 units per mg) was from Sigma, Heidelberg. Human chorionic gonadotrophin (hCG) was donated by Schering AG, Berlin. hCG for iodination was obtained from Calbiochem, San Diego (biological potency: ¡5 000 IU per mg). Synthetic gonadotrophin releasing hormone (Gn-RH) was a gift from Hoechst AG, Frankfurt. The reagents for the radioimmunoassay of rat LH were provided by the NIAMDD, Rat Pituitary Distribution Program.
Methods Testosterone was measured by radioimmunoassay without chromatography, as de¬ scribed earlier (Pirke 1973). For the measurement of testosterone in plasma which also contained iodinated hCG the ethyl ether extract was dried with NajSO-i in order to remove the small amount of the water contained in the ether phase. The inter-assay variability was 7% at an average concentration of 579 ng/100 ml. LH was measured by radioimmunoassay according to the recommendations of the Rat Pituitary Hormone Distribution Program. The results were expressed as ng LH-RP-1/ml. The intra-assay variability was ¡¡.5% (C.V.) at an average concentration of 42.4 ng/ml. In the pituitary gland LH was measured in the following way: The whole gland was homogenized in an all-glass Potter homogenizer in 1 ml isotonic phosphate buffer pH 7.4. The homogenate was then diluted ¡ :200 in the same buffer. One hundred «1 was used in the assay.
LH-RH-radioimmunoassay was coupled to bovine thyroglobulin (bTg) using bis-diazotized al. 1972). Approximately 48 molecules of LH-RH were coupled per one molecule of bTg. White female New Zealand rabbits were immunized according to the multiple site technique described by Vaitukailis et al. (1971). Ten µg of the conjugate was injected per animal. After 1 and 2 months booster injections were performed in the same way. After 3 months all rabbits had developed antisera. Five hundred «1 of the best serum diluted 1:20 000 bound 50 °/o of 50 pg iodinated LH-RH. Cross-reactivity of ACTH, «-MSH, human //-MSH, vasopressin and of 20 synthetic analogues kindly provided by Dr. Sandow, Hoechst AG, Frankfurt, was tested. All substances had less than 0.1 °/o cross-reactivity.
Synthetic LH-RH (Senyk et
benzidine
Iodination of LH-RH Five ,«g of LH-RH was dissolved in 100 µ 0.5 M phosphate buffer pH 7.4. One mCi 30 ¡tg cloramin-T and 30 fig Na¿SL)0;í were added. After 30 seconds at room temperature the mixture was chromatographed on a Sephadex G-15 column (10x0.5 cm). The iodinated hormone (S.A. 120-150 µC Iµg) was eluted well separated after the iodinepeak.
1251,
Assay was homogenized in 2 ml methanol. After centrifugation the dried down in a water bath (40°C) under a stream of nitrogen. The residue was re-dissolved in 500 til isotonic phosphate buffer. Two times 100 µ was used in the assay. Five hundred «1 of the 1:20 000 diluted antiserum and 100 µ of a 0.1 m EDTA solution were added. Normal rabbit serum (NRS) was added to give a final concentration of 0.5 °/o NRS. After 24 h at 4°C 8000 cpm of the freshly iodinated LH-RH were added in 100 nl buffer. On the third day the second antibody was added and the separation of bound and free was achieved by centrifugation on the fourth day. The precipitate was counted. The standard curve was prepared with 10, 25, 50, 100, 250, 500 and 1000 pg. The calculations were done using the logit-Iog plot (Rodbard el al. 1969). The intra-assay variability was 9.5 °/o at an average concentration of 150 pg/100 /(I.
Each
hypothalamus
supernatant
was
1: in vitro stimulation of isolated Leydig celles with hCG Immediately after sacrificing the rats both testes were removed, weighed and the external capsula was cut away. Both testes were incubated together in 2 ml isotonic phosphate buffer containing 2 mg collagenase. After 30 min of mixing, 15 ml buffer was added and after a short gentle mixing the plastic vials were left for 5 min. The supernatant was then taken off and again 5 ml buffer was added. The second super¬ natant was also removed after 5 min and the combined supernatant was centrifuged for 5 min at 200 g. The precipitate was re-suspended in 25 ml TC Medium 199 with Earle's salts. Five hundred ill of the cell suspension containing approximately 100 000 Leydig cells were incubated in plastic tubes after addition of 100 µ of a buffer solution containing 10 mg bovine serum albumin per ml and with either 10 IU of hCG or no hCG. The incubation was done in a metabolic shaker at 33°C under carbogen (95°/o 02, 5% COä). The incubation lasted for 30, 60, 90, 120, 180, 240 and 300 min. After removal from the incubator the samples were snap frozen in acetone dry ice and stored at 30°C until analysed. In order to evaluate the number of Leydig cells
Experiment
-
was incubated with 100 «1 nitroblue (2 mg/ml), NAD and 20 «1 dehydroepiandrosterone (1 mg/ml) for 3 h at 37°C. The Leydig cells which were thus stained dark blue were then counted in a Neubauer counting chamber under the microscope.
incubated, ¡00 µ of the cell suspension 100
,«1
2: in vivo stimulation of young and old rats by iv injection of iodinated biologically active hCG Highly purified hCG (15 000 IU/mg) was iodinated by the lactoperoxidase technique as described earlier in detail (Pirke et al. ¡978«). The specific activity was between ¡2 and 20 .«Ci/«g. The rats were lightly anaestetized with ethyl ether. One hundred µ oí the iodinated hCG in isotonic buffer was injected into the tail vein of the young rats and 140 µ into the old rats. These doses contained 2.95 ¡0fi cpm (0.27 ng 3.9 IU) or 4.13 cpm (0.37 ng 5.5 IU), respectively. The doses were chosen because pilot experi¬ ments had revealed that they were maximal doses with regard to the testosterone secretion and resulted in equal plasma levels of hCG in young and old animals. Control injections were done with isotonic saline. The rats were sacrificed by de¬ capitation after ¡5, 30, 60, ¡20, 180 and 240 min. The trunk blood was collected, testes, liver and brain were removed. For each point of time ¡2 old and 12 young rats were used. Six were injected with hCG and 6 served as controls. Five hundred mg of each organ was homogenized in 2 ml isotonic buffer in an all-glass Potter homogenizer. The homogenate was then diluted to a final volume of ¡0 ml, briefly mixed and centriluged at 13 000 g. The radioactivity of the precipitate was counted and expressed as
Experiment
=
=
counts per 100 mg wet tissue
weight.
Experiment
2a: influence of receptor capacity on gonadotrophin uptake The LH receptor capacity was artificially decreased in 3-month old rats by "desensitisation" (Usiteli et al. 1976; Diifaii et al. 1978). Groups of ¡0 rats were given a single sc injection of 10, 25,250 IU of hCG or of isotonic saline. On the fourth day half of the animals in each group were sacrificed and the LH receptor capacity was determined as described earlier (Pirke et al. ¡978«). The other half of each group was iv injected with one IU of iodinated hCG as described for experiment 2. After 2 h the rats were decapitated, blood was collected and the radioactivity bound in the testes was determined as described for experiment 2. 3: repeated stimulation of young and old rats with hCG Thirteen old and 14 young rats were injected subcutaneously with 50 IU hCG per day for 2 days. The rats were decapitated on the third day. Blood was collected and testosterone was measured as described.
Experiment
RESULTS
Table 1 shows the results obtained in 18 3-month old and in 19 24-month old Wistar rats. LH-RH in the hypothalamus, LH in the pituitary gland and LH and testosterone in plasma were all significantly lower in the old animals. The statistical comparison of old and young animals was done with the Wilcoxon test. The results of the in vitro stimulation of isolated Leydig cells
Table J. LH-RH in the
hypothalamus, LH in the pituitary gland and in serum, and testosterone in young adult and old male Wistar rats. LH-RH in the
LH in the
LHin
hypothalamus
pituitary gland (ng)
serum
(Pg)
X
±
SD
testosterone
in
(ng/ml) X
±
serum
X
SD
serum
ng/100 ml ±
SD
young adult rats
0=19)
581 ± 133
1707 ±278
63 ± 27
302 ± 198
394 ± 85
1272 ±219
31 ±11
108+ 53
old rats
(n=18)
o Q
— 60
-1-1-1-1-1-1180 120 240 300 t
Fig. 1.
(mm )
Testosterone production of isolated Leydig cells in vitro. The testosterone production of unstimulated Leydig cells is indicated by for the young and by for the old rats. The testosterone secretion under stimulation with 10 IU of hCG is indicated by · for the young and by O for the old rats. The vertical bars represent the standard deviations.
is illustrated in Fig. 1. The results of 6 experiments with 6 old and 6 young are summarized in the graph. The Leydig cells of both groups were stimulated with 10 IU of hCG The steroid production per Leydig cell is, however, significantly smaller in old age. The results of the second experiment are summarized in Fig. 2a-d. The concentrations of iodinated hCG in plasma (Fig. 2a) and the radioactivity in the precipitate from the liver homogenates (Fig. 2b) were not significantly different between young and old rats at any point in time Whereas radioactivity disappears rapidly from liver and plasma, rats
4 t
(h)
Fig. 2a. In vivo stimulation of young adult and old male Wistar rats by iv injection of bio¬ logically active iodinated hCG. The radioactivity in plasma of young and old rats. The vertical bats represent the standard error of the mean (Experiment 2).
en en
o o
4 t
(h)
Fig. 2b. Membrane bound
radioactivity
in the liver of young and old rats after
iodinated hCG
injection
of
(Experiment 2).
radioactivity is accumulated in the testes, thus indicating the specific bind¬ ing of the gonadotrophin in this organ. The testicular uptake of hCG was not significantly different in young and old animals (Fig. 2c). The increase of plasma testosterone, however, was significantly greater in the young rats (Fig. 2d).
the
artificially reduced LH uptake is not diminished when the receptor capacity is reduced by about 40%. Only when the remaining capacity dropped below 40 % was a reduced uptake of hCG observed. The
uptake
of iodinated hCG under conditions of
receptor capacity is indicated in Table 2. The
The results of the third experiment, in which 50 IU was injected for 2 days into young and old animals, demonstrate that chronic stimulation of young and old rats results in the same plasma testosterone levels. The old rats had testosterone levels between 650 and 1700 ng/100 ml (x 1086 ng/100 ml), while the young adult animals ranged between 700 and 2375 ng/ml (x 1071 ng/ =
=
100
ml). DISCUSSION
The decrease of testosterone and LH in plasma of old male Wistar rats listed in Table 1 confirms the data of Ghanadian et al. (1975) and of Chan et al. (1977) and are agreement with the data reported earlier by Pirke et al. (1978 ) in another group of young and old Wistar rats. We have described an increased
3 cn
en
3 cj
4 t
Fig. 2c.
(h)
Membrane bound radioactivity in testicular tissue of young and old rats after injection of iodinated hCG Vertical bars indicate the standard error of the mean (Experiment 2).
2 000
o
—
-1-1-1-1-* 3 4
12
(h)
Fig.
2d.
of young · and old O rats after iv injection of iodinated hCG Control and old animals were injected with saline. The young (Experiment 2). vertical bars indicate the standard error of the mean. Plasma testosterone was signi¬ ficantly higher in the young animals 1, 2 and 3 h after stimulation. < 0.05'\ < 0.02**, < 0.01***.
Testosterone in
plasma
sensitivity of the negative feedback testosterone-LH in old male rats (Pirke et al. 19786). It is, however, well known that negative feedback may be exerted at the pituitary as well as on at the hypothalamic level (Debcljuk et al. 1972; Cheung Sc Davidson 1977). Peng et al. (1973). using a bioassay, did not find
differences in LH-RH content of the median eminence of young and old male rats. Riegle et al. (1977), who also applied a bioassay to estimate the hypo¬ thalamic LH-RH, found no differences in pituitary LH production in vitro when the whole extract of one young or one old hypothalamus was incubated with pituitary tissue. When the extract of a half hypothalamus was used for stimulation of the pituitary a significantly higher LH output was achieved by young hypothalami. Our data are in agreement with this observation and
indicate that the decreased LH secretion in old rats is a consequence of a hypothalamic and not of a pituitary disturbance. Whether the changes in noradrenalin and serotonin turnover in the hypo¬ thalamus observed by Riegle 8c Miller (1978) are responsible for the reduced LH-RH content of the hypothalamus is not yet clear. The testosterone production of Leydig cells isolated from testes of old rats is significantly smaller than that from young adult animals (Fig. 1). We have earlier reported (Pirke et al. 1978 ) that the in vitro production of testicular tissue pieces (50 mg) from young and old rats was not different after hCG stimulation. This discrepancy can be resolved by the observation that the Leydig cell number is increased in the old testes (Lätzen 8c Überberg 1973; Pirke et al. 1978 ). It can be assumed that pieces of testicular tissue of young and old rats produced equal amounts of testosterone in vitro because there was greater number of Leydig cells in the tissue pieces of the old animals. The functional capacity of the single Leydig cell is, as indicated in Fig. 1, greatly reduced. After a single iv injection of hCG the testosterone concentrations in plasma were indeed higher in the young adult animals as compared with the old ones. The radioactivity in the plasma of young and old rats was not different, in-
Table 2. Effects of injection of a single dose of hCG on LH receptor capacity, iodinated hCG in the testes and on plasma testosterone (Experiment
uptake 2a).
LH receptor
capacity
nghCG/fOOmg testes
4.¡9±0.70
2.41
±
1.40
1.61 ± 20
0.91
± 0.42
(100%)
(57.5%)
(38.4 %)
(21.7%)
362 ± 48.1
351 ± 56.3
257 ± 59.6
20¡ ± 15.4
1800 ±284
1200 ±148
Membrane bound
radioactivity (labelled hCG) cpm/100 mg tissue
Plasma testosterone
ng/100 ml
1191
±210
1090 ±97
of
dicating that the hCG doses injected into young and old animals were indeed equivalent. The comparison of the time course of radioactivity in the precipitate after homogenisation of liver and testes (Fig. 2) shows a characteristic differ¬ ence. While the activity in the liver decreases parellel to the decrease of plasma concentrations reflecting the excretion of the labelled hCG, the activity in the testes is increased due to the binding of hCG to LH receptors of the Leydig cells. The curve is not a straight line passing through the zero point of the graph probably because the non-specific binding to the membranes is higher shortly after injection when all tissues are flooded with large amounts of labelled hCG. All of our attempts to correct the data for non-specific binding by measuring the contamination of spleen, adrenals, brain or heart muscles failed to convert the time course of radiactivity in the testes into a straight line passing the zero points. Nevertheless, Fig. 2c clearly indicates that the uptake of the iodinated hCG by the receptors of the Leydig cells is not different in young and old rats. This means that the reduction of the receptor capacity in testes of old rats does not influence their ability to accumulate gonadotrophin from the blood. The experiment 2a, in which we artificially reduced the hCG binding capacity by destroying the receptors by desensitisation (Hsueh et al. 1976), explains why a reduction of receptor number by 40 °/o which on the average occurs in old rats (Pirke et al. 1978 ) does not influence the testicular uptake of hCG from the blood. This experiment indicates that more than 60 °/o of the LH receptors need to be destroyed before the uptake of gonadotrophin from the blood is impaired. Repeated injections of high doses of hCG gave equally elevated testosterone values in young and old rats. In conclusion, the decreased hypothalamic LH-RH content indicates that the age-dependent decrease of Leydig cell function may be caused by an impaired hypothalamic dysfunction. As a consequence, the Leydig cells are in a chronically understimulated state, which severely impairs their ability to respond to acute stimulation by LH.
ACKNOWLEDGMENTS We wish to thank Dr. Parlow and the NIAMDD for the supply of reagents for the rat LH radioimmunoassay. We acknowledge the skilful technical assistance of Ms Ursula Wötzcl and Ms Brigitte Kerker.
REFERENCES Chan S. W.. Lealhcm j. II. Se Esashi T.: Endocrinology 101 (1977) 128. Cheung C. Se Davidson f.: Endocrinology 100 (1977) 292. Debcljuk L., Arimura A. Se Schally .: Endocrinology 90 (1972) 585. Dufau M., Hsueh ., Cigorraga S., Baukal A. Se Cult K.: Int. J. Androl. 193.
Suppl.
2
(1978)
Ghanadiiin R., Lewis J. 8c Chisholm G.: Steroids 25 (1975) 753. Hsueh ., Dufau M. 8- Catt K.: Biochem. biophys. Res. Commun. 72 (¡976) ¡145. Lätzen L. 8c Überberg H.: Beitr. path. Anat. Bd. 7-7.9 (1973) 495. Peng M., Pi W. Se Peng Y.: J. Formosan med. Ass. 72 (1973) 495. Pirke K. M.: Acta endocr. (Kbh.) 74 (1973) ¡68. Pirke K. M., Vogt H.-]. Se Geiss M.: Acta endocr. (Kbh.) 89 (1978«) 393. Pirke K. M., Geiss M. Se Sintermann R.: Acta endocr. (Kbh.) 89 (1978*) 789. Riegle G. Se Mettes J.: Proc. Soc. exp. Biol. (N. Y.) 757 (¡976) 507. Riegle G., Mettes J'., Miller A. Se Wood S.: J. Geront. 32 (¡977) ¡3. Riegle G. Se Miller A. In: Schneider E., Ed. The Aging Reproductive System (Aging, Vol. 4). Raven Press, New York (1978). Rodbard D., Bridson W. E. Se Rayford P. L.: J. Lab. clin. Med. 7-7 (¡969) 770. Senyk G., Nitecki D., Spitler L. Se Goodman J.: Immunochemistry 9 (¡972) 97. Shaar C. J., Euker f. S., Riegle G. D. Se Mettes J.: J. Endocr. 66 (¡975) 45. Vaitukaitis J., Robbins J., Nieschlag E. Se Ross G.: J. clin. Endocr. 33 (1971) 988. Received
on
January 29th,
1979.
FURTHER STUDIES ON HYPOTHALAMIC-PITUITARY-TESTICULAR FUNCTION IN OLD RATS
By Karl M. Pirke, Beate
Krings
and
Hermann-J. Vogt
ABSTRACT
dysfunction of the hypothalamic-pituitary-gonadal axis in old age studied in 24-month old male Wistar rats which were compared with 3-month old animals. The hypothalamic LH-RH content and the pituitary LH were significantly lower in the old than in the young adult animals. The plasma concentrations of LH and testosterone were significantly higher in the young rats. The primary cause of these age-dependent changes probably is a hypothalamic dysfunction. When isolated Leydig cells of young and old rats were incubated in vitro, the testosterone secretion per cell was significantly smaller in old than in young cells with as well as without HCG stimulation. In vivo stimulation of rats by iv injection of biologically active iodinated hCG revealed that the intratesticular uptake of the gonadotrophin was not different in young and old rats. The testosterone response, however, was significantly reduced in old age. An in vitro "desensitisation" experiment in which the LH receptor capacity was artificially reduced demonstrated that the 40 % reduction of receptor capacity in old testes as described earlier will not impair the testicular uptake of gonadotrophin from blood. Repeated injection of hCG results in equally elevated testosterone concentrations in young and old rats. The was
A decrease in
has been observed in the aging male rat Chan al. et 1977; Pirke et al. 1978 ). Although an age (Ghanadian et al. 1975; dependent fall in plasma LH indicates that the decline of the Leydig cell function is mainly caused by a pituitary or hypothalamic malfunction (Shaar et al 1975; Riegle 8c Mettes 1976; Pirke et al. 19786), other mechanisms may also be involved in reducing the testosterone production. We have recently
plasma
testosterone
described (Pirke et al. 1978 ) that the number of LH receptors as determined in vitro is significantly decreased in the testes of old rats. As a consequence, the testicular binding of LH and its effect on the Leydig cells may be altered in old animals. We have therefore studied the testicular response to stimulation by gonadotrophins in greater detail. We have examined the effect of hCG on isolated Leydig cells of young and old rats in vitro and the in vivo response to a single iv injection of HCG. We used iodinated hCG, which enabled us to follow the uptake and the binding of the gonadotrophic hormone. We have further studied the influence of repeated hCG injection into young adult and old rats.
MATERIALS AND METHODS
Animals Three-month old and 24-month old male Wistar rats were obtained from the Central Institute for Laboratory Animals, Hannover, Germany. The weight of the young rats ranged from 285 to 350 g (average: 3¡7 g). The average weight of the old rats was 435 g (range: 350 to 550 g). After the experiments all rats were studied by autopsy. All animals with atrophy or carcinoma of the testes or with other signs of severe illness were excluded from the studies. Four to 6 rats were housed in a cage under an 8-h dark, ¡6-h light schedule. Altrumine rat food and water were available ad libitum. Unless otherwise stated, the rats were sacrificed between 8:00 and ¡0:00 by decapita¬ tion immediately after removal from their cages. Trunk blood was collected. The hypothalamus and the pituitary gland were removed and snap frozen in acetone dry ice. Plasma and organs were stored at 30°C until analysed. -
Materials All reagents
were of analytical grade and obtained from Merck, Darmstadt unless otherwise stated. Collagenase (Typ III, 720 units per mg) was from Sigma, Heidelberg. Human chorionic gonadotrophin (hCG) was donated by Schering AG, Berlin. hCG for iodination was obtained from Calbiochem, San Diego (biological potency: ¡5 000 IU per mg). Synthetic gonadotrophin releasing hormone (Gn-RH) was a gift from Hoechst AG, Frankfurt. The reagents for the radioimmunoassay of rat LH were provided by the NIAMDD, Rat Pituitary Distribution Program.
Methods Testosterone was measured by radioimmunoassay without chromatography, as de¬ scribed earlier (Pirke 1973). For the measurement of testosterone in plasma which also contained iodinated hCG the ethyl ether extract was dried with NajSO-i in order to remove the small amount of the water contained in the ether phase. The inter-assay variability was 7% at an average concentration of 579 ng/100 ml. LH was measured by radioimmunoassay according to the recommendations of the Rat Pituitary Hormone Distribution Program. The results were expressed as ng LH-RP-1/ml. The intra-assay variability was ¡¡.5% (C.V.) at an average concentration of 42.4 ng/ml. In the pituitary gland LH was measured in the following way: The whole gland was homogenized in an all-glass Potter homogenizer in 1 ml isotonic phosphate buffer pH 7.4. The homogenate was then diluted ¡ :200 in the same buffer. One hundred «1 was used in the assay.
LH-RH-radioimmunoassay was coupled to bovine thyroglobulin (bTg) using bis-diazotized al. 1972). Approximately 48 molecules of LH-RH were coupled per one molecule of bTg. White female New Zealand rabbits were immunized according to the multiple site technique described by Vaitukailis et al. (1971). Ten µg of the conjugate was injected per animal. After 1 and 2 months booster injections were performed in the same way. After 3 months all rabbits had developed antisera. Five hundred «1 of the best serum diluted 1:20 000 bound 50 °/o of 50 pg iodinated LH-RH. Cross-reactivity of ACTH, «-MSH, human //-MSH, vasopressin and of 20 synthetic analogues kindly provided by Dr. Sandow, Hoechst AG, Frankfurt, was tested. All substances had less than 0.1 °/o cross-reactivity.
Synthetic LH-RH (Senyk et
benzidine
Iodination of LH-RH Five ,«g of LH-RH was dissolved in 100 µ 0.5 M phosphate buffer pH 7.4. One mCi 30 ¡tg cloramin-T and 30 fig Na¿SL)0;í were added. After 30 seconds at room temperature the mixture was chromatographed on a Sephadex G-15 column (10x0.5 cm). The iodinated hormone (S.A. 120-150 µC Iµg) was eluted well separated after the iodinepeak.
1251,
Assay was homogenized in 2 ml methanol. After centrifugation the dried down in a water bath (40°C) under a stream of nitrogen. The residue was re-dissolved in 500 til isotonic phosphate buffer. Two times 100 µ was used in the assay. Five hundred «1 of the 1:20 000 diluted antiserum and 100 µ of a 0.1 m EDTA solution were added. Normal rabbit serum (NRS) was added to give a final concentration of 0.5 °/o NRS. After 24 h at 4°C 8000 cpm of the freshly iodinated LH-RH were added in 100 nl buffer. On the third day the second antibody was added and the separation of bound and free was achieved by centrifugation on the fourth day. The precipitate was counted. The standard curve was prepared with 10, 25, 50, 100, 250, 500 and 1000 pg. The calculations were done using the logit-Iog plot (Rodbard el al. 1969). The intra-assay variability was 9.5 °/o at an average concentration of 150 pg/100 /(I.
Each
hypothalamus
supernatant
was
1: in vitro stimulation of isolated Leydig celles with hCG Immediately after sacrificing the rats both testes were removed, weighed and the external capsula was cut away. Both testes were incubated together in 2 ml isotonic phosphate buffer containing 2 mg collagenase. After 30 min of mixing, 15 ml buffer was added and after a short gentle mixing the plastic vials were left for 5 min. The supernatant was then taken off and again 5 ml buffer was added. The second super¬ natant was also removed after 5 min and the combined supernatant was centrifuged for 5 min at 200 g. The precipitate was re-suspended in 25 ml TC Medium 199 with Earle's salts. Five hundred ill of the cell suspension containing approximately 100 000 Leydig cells were incubated in plastic tubes after addition of 100 µ of a buffer solution containing 10 mg bovine serum albumin per ml and with either 10 IU of hCG or no hCG. The incubation was done in a metabolic shaker at 33°C under carbogen (95°/o 02, 5% COä). The incubation lasted for 30, 60, 90, 120, 180, 240 and 300 min. After removal from the incubator the samples were snap frozen in acetone dry ice and stored at 30°C until analysed. In order to evaluate the number of Leydig cells
Experiment
-
was incubated with 100 «1 nitroblue (2 mg/ml), NAD and 20 «1 dehydroepiandrosterone (1 mg/ml) for 3 h at 37°C. The Leydig cells which were thus stained dark blue were then counted in a Neubauer counting chamber under the microscope.
incubated, ¡00 µ of the cell suspension 100
,«1
2: in vivo stimulation of young and old rats by iv injection of iodinated biologically active hCG Highly purified hCG (15 000 IU/mg) was iodinated by the lactoperoxidase technique as described earlier in detail (Pirke et al. ¡978«). The specific activity was between ¡2 and 20 .«Ci/«g. The rats were lightly anaestetized with ethyl ether. One hundred µ oí the iodinated hCG in isotonic buffer was injected into the tail vein of the young rats and 140 µ into the old rats. These doses contained 2.95 ¡0fi cpm (0.27 ng 3.9 IU) or 4.13 cpm (0.37 ng 5.5 IU), respectively. The doses were chosen because pilot experi¬ ments had revealed that they were maximal doses with regard to the testosterone secretion and resulted in equal plasma levels of hCG in young and old animals. Control injections were done with isotonic saline. The rats were sacrificed by de¬ capitation after ¡5, 30, 60, ¡20, 180 and 240 min. The trunk blood was collected, testes, liver and brain were removed. For each point of time ¡2 old and 12 young rats were used. Six were injected with hCG and 6 served as controls. Five hundred mg of each organ was homogenized in 2 ml isotonic buffer in an all-glass Potter homogenizer. The homogenate was then diluted to a final volume of ¡0 ml, briefly mixed and centriluged at 13 000 g. The radioactivity of the precipitate was counted and expressed as
Experiment
=
=
counts per 100 mg wet tissue
weight.
Experiment
2a: influence of receptor capacity on gonadotrophin uptake The LH receptor capacity was artificially decreased in 3-month old rats by "desensitisation" (Usiteli et al. 1976; Diifaii et al. 1978). Groups of ¡0 rats were given a single sc injection of 10, 25,250 IU of hCG or of isotonic saline. On the fourth day half of the animals in each group were sacrificed and the LH receptor capacity was determined as described earlier (Pirke et al. ¡978«). The other half of each group was iv injected with one IU of iodinated hCG as described for experiment 2. After 2 h the rats were decapitated, blood was collected and the radioactivity bound in the testes was determined as described for experiment 2. 3: repeated stimulation of young and old rats with hCG Thirteen old and 14 young rats were injected subcutaneously with 50 IU hCG per day for 2 days. The rats were decapitated on the third day. Blood was collected and testosterone was measured as described.
Experiment
RESULTS
Table 1 shows the results obtained in 18 3-month old and in 19 24-month old Wistar rats. LH-RH in the hypothalamus, LH in the pituitary gland and LH and testosterone in plasma were all significantly lower in the old animals. The statistical comparison of old and young animals was done with the Wilcoxon test. The results of the in vitro stimulation of isolated Leydig cells
Table J. LH-RH in the
hypothalamus, LH in the pituitary gland and in serum, and testosterone in young adult and old male Wistar rats. LH-RH in the
LH in the
LHin
hypothalamus
pituitary gland (ng)
serum
(Pg)
X
±
SD
testosterone
in
(ng/ml) X
±
serum
X
SD
serum
ng/100 ml ±
SD
young adult rats
0=19)
581 ± 133
1707 ±278
63 ± 27
302 ± 198
394 ± 85
1272 ±219
31 ±11
108+ 53
old rats
(n=18)
o Q
— 60
-1-1-1-1-1-1180 120 240 300 t
Fig. 1.
(mm )
Testosterone production of isolated Leydig cells in vitro. The testosterone production of unstimulated Leydig cells is indicated by for the young and by for the old rats. The testosterone secretion under stimulation with 10 IU of hCG is indicated by · for the young and by O for the old rats. The vertical bars represent the standard deviations.
is illustrated in Fig. 1. The results of 6 experiments with 6 old and 6 young are summarized in the graph. The Leydig cells of both groups were stimulated with 10 IU of hCG The steroid production per Leydig cell is, however, significantly smaller in old age. The results of the second experiment are summarized in Fig. 2a-d. The concentrations of iodinated hCG in plasma (Fig. 2a) and the radioactivity in the precipitate from the liver homogenates (Fig. 2b) were not significantly different between young and old rats at any point in time Whereas radioactivity disappears rapidly from liver and plasma, rats
4 t
(h)
Fig. 2a. In vivo stimulation of young adult and old male Wistar rats by iv injection of bio¬ logically active iodinated hCG. The radioactivity in plasma of young and old rats. The vertical bats represent the standard error of the mean (Experiment 2).
en en
o o
4 t
(h)
Fig. 2b. Membrane bound
radioactivity
in the liver of young and old rats after
iodinated hCG
injection
of
(Experiment 2).
radioactivity is accumulated in the testes, thus indicating the specific bind¬ ing of the gonadotrophin in this organ. The testicular uptake of hCG was not significantly different in young and old animals (Fig. 2c). The increase of plasma testosterone, however, was significantly greater in the young rats (Fig. 2d).
the
artificially reduced LH uptake is not diminished when the receptor capacity is reduced by about 40%. Only when the remaining capacity dropped below 40 % was a reduced uptake of hCG observed. The
uptake
of iodinated hCG under conditions of
receptor capacity is indicated in Table 2. The
The results of the third experiment, in which 50 IU was injected for 2 days into young and old animals, demonstrate that chronic stimulation of young and old rats results in the same plasma testosterone levels. The old rats had testosterone levels between 650 and 1700 ng/100 ml (x 1086 ng/100 ml), while the young adult animals ranged between 700 and 2375 ng/ml (x 1071 ng/ =
=
100
ml). DISCUSSION
The decrease of testosterone and LH in plasma of old male Wistar rats listed in Table 1 confirms the data of Ghanadian et al. (1975) and of Chan et al. (1977) and are agreement with the data reported earlier by Pirke et al. (1978 ) in another group of young and old Wistar rats. We have described an increased
3 cn
en
3 cj
4 t
Fig. 2c.
(h)
Membrane bound radioactivity in testicular tissue of young and old rats after injection of iodinated hCG Vertical bars indicate the standard error of the mean (Experiment 2).
2 000
o
—
-1-1-1-1-* 3 4
12
(h)
Fig.
2d.
of young · and old O rats after iv injection of iodinated hCG Control and old animals were injected with saline. The young (Experiment 2). vertical bars indicate the standard error of the mean. Plasma testosterone was signi¬ ficantly higher in the young animals 1, 2 and 3 h after stimulation. < 0.05'\ < 0.02**, < 0.01***.
Testosterone in
plasma
sensitivity of the negative feedback testosterone-LH in old male rats (Pirke et al. 19786). It is, however, well known that negative feedback may be exerted at the pituitary as well as on at the hypothalamic level (Debcljuk et al. 1972; Cheung Sc Davidson 1977). Peng et al. (1973). using a bioassay, did not find
differences in LH-RH content of the median eminence of young and old male rats. Riegle et al. (1977), who also applied a bioassay to estimate the hypo¬ thalamic LH-RH, found no differences in pituitary LH production in vitro when the whole extract of one young or one old hypothalamus was incubated with pituitary tissue. When the extract of a half hypothalamus was used for stimulation of the pituitary a significantly higher LH output was achieved by young hypothalami. Our data are in agreement with this observation and
indicate that the decreased LH secretion in old rats is a consequence of a hypothalamic and not of a pituitary disturbance. Whether the changes in noradrenalin and serotonin turnover in the hypo¬ thalamus observed by Riegle 8c Miller (1978) are responsible for the reduced LH-RH content of the hypothalamus is not yet clear. The testosterone production of Leydig cells isolated from testes of old rats is significantly smaller than that from young adult animals (Fig. 1). We have earlier reported (Pirke et al. 1978 ) that the in vitro production of testicular tissue pieces (50 mg) from young and old rats was not different after hCG stimulation. This discrepancy can be resolved by the observation that the Leydig cell number is increased in the old testes (Lätzen 8c Überberg 1973; Pirke et al. 1978 ). It can be assumed that pieces of testicular tissue of young and old rats produced equal amounts of testosterone in vitro because there was greater number of Leydig cells in the tissue pieces of the old animals. The functional capacity of the single Leydig cell is, as indicated in Fig. 1, greatly reduced. After a single iv injection of hCG the testosterone concentrations in plasma were indeed higher in the young adult animals as compared with the old ones. The radioactivity in the plasma of young and old rats was not different, in-
Table 2. Effects of injection of a single dose of hCG on LH receptor capacity, iodinated hCG in the testes and on plasma testosterone (Experiment
uptake 2a).
LH receptor
capacity
nghCG/fOOmg testes
4.¡9±0.70
2.41
±
1.40
1.61 ± 20
0.91
± 0.42
(100%)
(57.5%)
(38.4 %)
(21.7%)
362 ± 48.1
351 ± 56.3
257 ± 59.6
20¡ ± 15.4
1800 ±284
1200 ±148
Membrane bound
radioactivity (labelled hCG) cpm/100 mg tissue
Plasma testosterone
ng/100 ml
1191
±210
1090 ±97
of
dicating that the hCG doses injected into young and old animals were indeed equivalent. The comparison of the time course of radioactivity in the precipitate after homogenisation of liver and testes (Fig. 2) shows a characteristic differ¬ ence. While the activity in the liver decreases parellel to the decrease of plasma concentrations reflecting the excretion of the labelled hCG, the activity in the testes is increased due to the binding of hCG to LH receptors of the Leydig cells. The curve is not a straight line passing through the zero point of the graph probably because the non-specific binding to the membranes is higher shortly after injection when all tissues are flooded with large amounts of labelled hCG. All of our attempts to correct the data for non-specific binding by measuring the contamination of spleen, adrenals, brain or heart muscles failed to convert the time course of radiactivity in the testes into a straight line passing the zero points. Nevertheless, Fig. 2c clearly indicates that the uptake of the iodinated hCG by the receptors of the Leydig cells is not different in young and old rats. This means that the reduction of the receptor capacity in testes of old rats does not influence their ability to accumulate gonadotrophin from the blood. The experiment 2a, in which we artificially reduced the hCG binding capacity by destroying the receptors by desensitisation (Hsueh et al. 1976), explains why a reduction of receptor number by 40 °/o which on the average occurs in old rats (Pirke et al. 1978 ) does not influence the testicular uptake of hCG from the blood. This experiment indicates that more than 60 °/o of the LH receptors need to be destroyed before the uptake of gonadotrophin from the blood is impaired. Repeated injections of high doses of hCG gave equally elevated testosterone values in young and old rats. In conclusion, the decreased hypothalamic LH-RH content indicates that the age-dependent decrease of Leydig cell function may be caused by an impaired hypothalamic dysfunction. As a consequence, the Leydig cells are in a chronically understimulated state, which severely impairs their ability to respond to acute stimulation by LH.
ACKNOWLEDGMENTS We wish to thank Dr. Parlow and the NIAMDD for the supply of reagents for the rat LH radioimmunoassay. We acknowledge the skilful technical assistance of Ms Ursula Wötzcl and Ms Brigitte Kerker.
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2
(1978)
Ghanadiiin R., Lewis J. 8c Chisholm G.: Steroids 25 (1975) 753. Hsueh ., Dufau M. 8- Catt K.: Biochem. biophys. Res. Commun. 72 (¡976) ¡145. Lätzen L. 8c Überberg H.: Beitr. path. Anat. Bd. 7-7.9 (1973) 495. Peng M., Pi W. Se Peng Y.: J. Formosan med. Ass. 72 (1973) 495. Pirke K. M.: Acta endocr. (Kbh.) 74 (1973) ¡68. Pirke K. M., Vogt H.-]. Se Geiss M.: Acta endocr. (Kbh.) 89 (1978«) 393. Pirke K. M., Geiss M. Se Sintermann R.: Acta endocr. (Kbh.) 89 (1978*) 789. Riegle G. Se Mettes J.: Proc. Soc. exp. Biol. (N. Y.) 757 (¡976) 507. Riegle G., Mettes J'., Miller A. Se Wood S.: J. Geront. 32 (¡977) ¡3. Riegle G. Se Miller A. In: Schneider E., Ed. The Aging Reproductive System (Aging, Vol. 4). Raven Press, New York (1978). Rodbard D., Bridson W. E. Se Rayford P. L.: J. Lab. clin. Med. 7-7 (¡969) 770. Senyk G., Nitecki D., Spitler L. Se Goodman J.: Immunochemistry 9 (¡972) 97. Shaar C. J., Euker f. S., Riegle G. D. Se Mettes J.: J. Endocr. 66 (¡975) 45. Vaitukaitis J., Robbins J., Nieschlag E. Se Ross G.: J. clin. Endocr. 33 (1971) 988. Received
on
January 29th,
1979.
