0013-7227/90/1275-2469$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Differential Response of Luteinizing Hormone Receptor and Steroidogenic Enzyme Gene Expression to Human Chorionic Gonadotropin Stimulation in the Neonatal and Adult Rat Testis* PIRJO PAKARINEN, KIMMO K. VIHKO, RAIMO VOUTILAINEN, AND ILPO HUHTANIEMI Department of Physiology, University of Turku, SF-20520 Turku, and Departments of Pathology (R. V.) and Pediatrics (R. V.), University of Helsinki, SF-00290 Helsinki, Finland
ABSTRACT. To study further the functional differences of the fetal-neonatal and adult growth phases of Leydig cells, neonatal (5-day-old) and adult (60-day-old) male rats were challenged with a 600 IU/kg injection of human CG (hCG). Certain Leydig cell responses were monitored 1, 2, and 3 days after the hCG injection. The down-regulation of LH receptors and blockage of the 17-hydroxylase/Cl7-20 lyase step in adult testis, and the absence of these responses in neonatal testis were confirmed. Novel data were obtained on concomitant responses of LH receptor and steroidogenic enzyme messenger RNAs (mRNAs). The LH receptor mRNA was increased 4-5-fold by 2 days after hCG injection in the neonatal testis (P < 0.05), but in the adult was decreased during all 3 days by 50% (P < 0.05). The mRNA level of the cytochrome P450 for cholesterol side chain cleavage responded similarly at both ages, with a 180-260% increase during 2 and 3 days (P < 0.05-0.01). In contrast, the 17hydroxylase/17,20-lyase cytochrome P450 mRNAs displayed op-
D
URING development, Leydig cells can be divided into two functionally distinct growth phases, termed the fetal and adult populations (1-3). The fetal population appears in utero and is gradually replaced by the adult population during pubertal maturation. The two populations function in endocrinologically different environments, because the fetal population is exposed to the high levels of placental protein and steroid hormones. Both high gonadotropin and estrogen levels are known to be inhibitory to adult testicular function including LH receptor down-regulation and blockade of steroidogenesis at the 17-hydroxylase/Cl7-20 lyase step (i.e. steroidogenic lesion) (4-6). If the regulation of the fetal testes Received April 9, 1990. Address all correspondence and requests for reprints to: Dr. Pirjo Pakarinen, Department of Physiology, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland. * These studies were supported by research contracts from the Academy of Finland and the Finnish Life and Pension Insurance Companies, and by a grant from the Sigrid Juselius Foundation.
posite responses, increasing 4.5-fold in 2 days (P < 0.01) in the neonates, but decreasing by 80% in 1 day in the adults (P < 0.01). No response of the aromatase cytochrome P450 mRNA to hCG stimulation was found at either age studied. These results demonstrate that the functional differences of the neonatal and adult Leydig cells to high gonadotropic stimulation occur at the level of expression of specific genes, including those of the LH receptor and the 17-hydroxylase/17,20-lyase cytochrome P450. Although aromatization of testicular androgens has been suggested to mediate the blockade of the 17-hydroxylase/Cl7-20 lyase step in adult testes, altered steady state levels of aromatase mRNA are not involved in this response. LH receptor mRNA decreases in adult rat testis in response to treatment with high levels of hCG. Thus, this phenomenon of down-regulation of membrane receptors includes a decreased LH receptor mRNA as well as cellular internalization of the existing receptors. (Endocrinology 127: 2469-2474, 1990)
were the same, they would be inhibited by the intrauterine hormone milieu and unable to produce the high levels of testosterone needed for male genital differentiation (7, 8). The fetal-neonatal and adult populations of Leydig cells respond differently to high gonadotropic stimulation (3, 9-11). After human CG (hCG) stimulation, the adult testis shows a distinct LH receptor down-regulation and steroidogenic lesion (4-6), but both of these negative responses are missing in the fetal and neonatal testes. Instead, hCG stimulation increases the testicular LH receptor content and capacity to produce testosterone (3, 9-11). The molecular mechanisms responsible for these functional differences have so far remained unclear. As an attempt to delineate these mechanisms we now have studied the effects of hCG treatment on neonatal and adult testis mRNA levels of the LH receptor and of key steroidogenic enzymes. Besides elucidating these mechanisms in the adult testes, we also show that
2469
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 19:18 For personal use only. No other uses without permission. . All rights reserved.
hCG STIMULATION IN TESTIS
2470
clear-cut differences prevail between neonatal and adult rats in the expression of the LH receptor and specific steroidogenic enzyme genes.
Materials and Methods A single dose of hCG (Pregnyl, Organon, Oss, The Netherlands), 600 IU/kg body wt, was injected sc into neonatal (5day-old) and adult (60-day-old) male rats of the SpragueDawley strain produced in our own vivarium. The dose selected was based on earlier dose-response studies on hCG effects on rat testicular LH receptors and steroidogenesis (9). The rats were killed by decapitation 1, 2, or 3 days after hCG treatment. Trunk blood was collected and the serum separated and stored at —20 C. The testes were removed and either immediately snap-frozen in liquid nitrogen and stored at -70 C, or immersed directly into 4 M guanidinium thiocyanate reagent solution (GIT, Eastman Kodak Company, Rochester, NY; 0.5% sodium lauroylsarcosine, Sigma Chemical Company, St. Louis, MO; 25 mM sodium citrate, Sigma; 0.7% 2-mercapto-ethanol, Sigma; 0.1% antifoam A, Sigma) and stored, after homogenization, at -20 C. Serum testosterone and progesterone concentrations were measured from diethyl ether extracts by RIA as described previously (12, 13). The testosterone assay sensitivity was 1 fmol/tube; the intraassay coefficient of variation was less than 6%, and the interassay coefficient of variation less than 12%. The progesterone assay sensitivity was 4 fmol/tube; the intraassay coefficient of variation was 10%; and the interassay coefficient of variation 15%. The LH receptor measurements were performed as described before (9, 14, 15). A solid-phase lactoperoxidase method (16) was used for radioiodination of highly purified hCG (CR-121, 13,500 IU/mg by bioassay, prepared by Dr. R. E. Canfield, Columbia University, New York, NY, and provided by National Institute for Child Health and Human Development, Bethesda, MD). Total RNA was isolated from the deep-frozen testes by the proteinase K method (17) and from the GIT solution by a CsCl centrifugation method (18). The mRNAs were analyzed by a cDNA hybridization assay in slot blots (Minifold II, Scheicher & Schuell GmbH, Dassel, West Germany). Total denatured RNA (2-10 jug) was immobilized in each slot in GeneScreen hybridization membranes (Du Pont, NEN Research Products, Boston, MA). The RNA was fixed by UV irradiation for 5 min. Prehybridization and hybridization were performed at 42 C as described previously (19). The cDNA inserts were labeled by a Multiprime DNA labeling system (Amersham International pic, Amersham, Bucks, UK) with a [a-32P]deoxycytidine 5'triphosphate (3000 Ci/mmol). The cDNAs used were those of rat aromatase cytochrome P450 (P450arom) (20), human cholesterol side chain cleavage cytochrome P450 (P450scc) (21) and human 17-hydroxylase/17,20-lyase cytochrome P450 (P450cl7) (22). For LH receptor measurements a 40-mer oligonucleotide (nucleotides 232-271 of the rat LH receptor cDNA) (23) was synthesized and labeled by a DNA 3'-End Labeling Kit (Boehringer Mannheim GmbH, Mannheim, West Germany) with a [a-32P]dCTP (3000 Ci/mmol). The washed filters were autoradiographed using x-ray film
Endo • 1990 Vol 127 • No 5
(Kodak XAR-5, Eastman Kodak Company, Rochester, NY) and intensifying screens for 3-21 days at —50 C. Autoradiograms were scanned by an LKB UltroScan XL 2222-020 densitometer, and an LKB 2400 GelScan XL software package (LKB-Produkter AB, Bromma, Sweden) was used for the calculation of results. The mean control levels in each experiment were assigned values of 100%. Hybridization with a-actin cDNA demonstrated that similar amounts of RNA were present in parallel slots of the same experiments. Because expression of specific cDNA hybridization per a-actin hybridization did not affect the results, this correction was omitted in calculation of the final results. Each cDNA and oligonucleotide probe was hybridized with Northern blots of rat gonadal RNA, and hybridization only occurred with the RNA species of correct molecular sizes, according to literature (21-23). No hybridization was observed with RNA prepared for nonendocrine control tissue (liver). All samples to be compared were immobilized on the same membrane. The data were analyzed by one-way analysis of variance and Duncan's new multiple range test. When comparing two means the unpaired Student's t test was used. A P value less than 0.05 was chosen as the limit of statistical significance.
Results Injection of 600 IU/kg hCG increased serum testosterone levels at both ages studies, about 3-fold (P < 0.01) in 2 days in the neonates, and from 7-12-fold (P < 0.01) between 1-3 days in the adults (Fig. 1, upper panel). The other serum steroid analyzed was progesterone, which showed no change in the neonates (Fig. 1, lower panel). In contrast, in the adult, the basal concentration of progesterone increased as much as 12-15-fold on days 1 and 2 of the experiment (P < 0.01). The mRNA level of testicular P450scc increased between 1-3 days by 80-130% (P < 0.05) in the neonates and between 80-170% (P < 0.05-0.01) in the adults (Fig. 2, upper panel). The responses of the P450cl7 mRNAs were opposite at the two ages studied, displaying a greater than 4-fold increase (P < 0.01) in the neonatal testes but a 75% decrease (P < 0.01) in the adults (Fig. 2, middle panel). No response was observed in P450arom mRNA at either age (Fig. 2, lower panel). The unoccupied LH receptors and the respective mRNA levels before and after hCG treatment are shown in Fig. 3. In both parameters measured opposite effects were observed between the two ages. The number of unoccupied LH receptors was very low 1 day after hCG injection. It recovered rapidly by day 3 in the neonates but remained undetectable on days 2 and 3 in the adult testes. A 4-5-fold increase (P < 0.05) in LH receptor mRNA was observed in the neonates on days 2 and 3, and a concomitant decrease of 50% (P < 0.05) occurred in the adult testes at all 3 days after treatment. Table 1 presents the relative concentrations of the different parameters measured in the neonatal and adult
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 19:18 For personal use only. No other uses without permission. . All rights reserved.
hCG STIMULATION IN TESTIS
5.0
-1..
100 ADULT
0.0
0
1 2
3
I.I
40
0
••
7
60
20
300
**
80
III
% of Controi;
^ 10.0
2471
i
-111 0
1
2
200
100
0
NEONATE
.III 0
3
300
1
2
ADULT
NEONATE
1
2
3
150 NEONATE
.§
j> 0.3
a.
0
••
ADULT
15
0.4
2
100
3
600
20 0.5
200
Days After hCG Injection
Days After hCG Injection
|
ADULT
400
100
200
50
8
10
0.2
2 0.1 4>
0.0 0
1
2
3
0
1
2
3
3
Days After hCG Injection
200
High-dose stimulation with LH/hCG results in loss of LH receptors and blockade of the 17-hydroxylase/Cl720 lyase step in adult rat Leydig cells (4, 24). It is not yet known to what extent increased sequestration, internalization, and intracellular degradation, and decreased receptor protein synthesis are responsible for the LH receptor down-regulation. There is considerable information that the steroidogenic blockade is an estrogen mediated phenomenon, where intratesticular aromatization of androgens and estrogen receptor mediated synthesis of a specific protein in Leydig cells play a role (24, 25). Previous studies have shown increased receptor internalization and degradation during down-regulation in
200 NEONATE
so
Discussion
1
Days After hCG Injection
FlG. 1. Serum testosterone (upper panel) and progesterone (lower panel) in the serum of neonatal (5-day-old, left) and adult (60-day-old, right) male rats before (day 0) and 1, 2, and 3 days after a 600 IU/kg body wt sc injection of hCG. Each bar is the mean ± SE of measurements from 5-12 animals. The asterisks indicate significant differences from the 0-day level (**, P < 0.01, Duncan's test).
rat testis tissue or serum. The basal serum concentrations of progesterone and testosterone were both 2-fold higher in the adult rat. No difference occurred between testicular concentrations of the cholesterol side chain cleavage and aromatase mRNA levels, but that of 17hydroxylase/17,20-lyase was 2.7-fold higher in the adult testis. The LH receptor mRNA concentration was 10fold higher in the adult testis.
0
100
ADULT
Illl llll 0
1
2
3 0 Days After hCG Injection
1
2
3
FIG. 2. Testicular concentrations of mRNAs for P450scc (upper panel), P450cl7 (middle panel) and P450 arom (lower panel) before and up to 3 days after a sc injection of 600 IU/kg hCG. ADU, arbitrary densitometric unit. Each bar is the mean ± SE of measurements from 5-12 animals. *, P < 0.05; **, P < 0.01 as compared with 0-day level.
adult testes (4). Our present data suggest that the rate of receptor synthesis also may be reduced, although they do not provide direct evidence whether the suppressed steady state level of LH receptor mRNA is due to decreased synthesis or increased degradation of the message. The loss of mRNA was less pronounced than that of the receptor protein. This is logical, because the actual receptor loss is the sum of several negative effects, i.e. increased receptor occupancy, increased receptor sequestration and intracellular degradation, decreased LH receptor gene transcription or increased mRNA degradation, and decreased protein synthesis. The LH receptor mRNA levels in fact only monitor the last responses. A decrease of receptor mRNA levels has been demonstrated
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 19:18 For personal use only. No other uses without permission. . All rights reserved.
hCG STIMULATION IN TESTIS
2472 1.5
0.15 NEONATE
0.10
1.0
0.05
0.5
0.00
0
1 2
ADULT
0.0
3
0
1 2
3
1 2
3
Days After hCG Injection
800 ADULT
NEONATE
600 -
100
400 50 200
0
1
2
3
0
Days After hCG Injection
FIG. 3. Testicular concentrations of LH receptors (upper panel) and the mRNA for LH receptor (lower panel) before and up to 3 days after a 600 IU/kg sc injection of hCG. ADU, arbitrary densitometric unit. Each bar is the mean ± SE of measurements from 5-12 animals. *, P < 0.05; **, P < 0.01 as compared with 0-day level. TABLE 1. Comparison between the neonatal and adult male rats of relative abundances of serum (S) progesterone and testosterone, and on testicular mRNAs of P450scc, P450cl7, P450arom, and LH receptor
S-progesterone S-testosterone P450scc P450cl7 P450arom LH receptor
Neonates
(n)
Adults
(n)
P value
100 ± 17 100 ± 27 100 ± 30 100 ± 27 100 ± 6 100 ± 36
(10) (7) (5) (5) (15) (10)
218 ± 64 209 ± 20 83 ±20 267 ± 33 80 ± 7 1075 ± 167
(5) (5) (5) (5) (14) (10)
Vol. 127, No. 5 Printed in U.S.A.
Differential Response of Luteinizing Hormone Receptor and Steroidogenic Enzyme Gene Expression to Human Chorionic Gonadotropin Stimulation in the Neonatal and Adult Rat Testis* PIRJO PAKARINEN, KIMMO K. VIHKO, RAIMO VOUTILAINEN, AND ILPO HUHTANIEMI Department of Physiology, University of Turku, SF-20520 Turku, and Departments of Pathology (R. V.) and Pediatrics (R. V.), University of Helsinki, SF-00290 Helsinki, Finland
ABSTRACT. To study further the functional differences of the fetal-neonatal and adult growth phases of Leydig cells, neonatal (5-day-old) and adult (60-day-old) male rats were challenged with a 600 IU/kg injection of human CG (hCG). Certain Leydig cell responses were monitored 1, 2, and 3 days after the hCG injection. The down-regulation of LH receptors and blockage of the 17-hydroxylase/Cl7-20 lyase step in adult testis, and the absence of these responses in neonatal testis were confirmed. Novel data were obtained on concomitant responses of LH receptor and steroidogenic enzyme messenger RNAs (mRNAs). The LH receptor mRNA was increased 4-5-fold by 2 days after hCG injection in the neonatal testis (P < 0.05), but in the adult was decreased during all 3 days by 50% (P < 0.05). The mRNA level of the cytochrome P450 for cholesterol side chain cleavage responded similarly at both ages, with a 180-260% increase during 2 and 3 days (P < 0.05-0.01). In contrast, the 17hydroxylase/17,20-lyase cytochrome P450 mRNAs displayed op-
D
URING development, Leydig cells can be divided into two functionally distinct growth phases, termed the fetal and adult populations (1-3). The fetal population appears in utero and is gradually replaced by the adult population during pubertal maturation. The two populations function in endocrinologically different environments, because the fetal population is exposed to the high levels of placental protein and steroid hormones. Both high gonadotropin and estrogen levels are known to be inhibitory to adult testicular function including LH receptor down-regulation and blockade of steroidogenesis at the 17-hydroxylase/Cl7-20 lyase step (i.e. steroidogenic lesion) (4-6). If the regulation of the fetal testes Received April 9, 1990. Address all correspondence and requests for reprints to: Dr. Pirjo Pakarinen, Department of Physiology, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland. * These studies were supported by research contracts from the Academy of Finland and the Finnish Life and Pension Insurance Companies, and by a grant from the Sigrid Juselius Foundation.
posite responses, increasing 4.5-fold in 2 days (P < 0.01) in the neonates, but decreasing by 80% in 1 day in the adults (P < 0.01). No response of the aromatase cytochrome P450 mRNA to hCG stimulation was found at either age studied. These results demonstrate that the functional differences of the neonatal and adult Leydig cells to high gonadotropic stimulation occur at the level of expression of specific genes, including those of the LH receptor and the 17-hydroxylase/17,20-lyase cytochrome P450. Although aromatization of testicular androgens has been suggested to mediate the blockade of the 17-hydroxylase/Cl7-20 lyase step in adult testes, altered steady state levels of aromatase mRNA are not involved in this response. LH receptor mRNA decreases in adult rat testis in response to treatment with high levels of hCG. Thus, this phenomenon of down-regulation of membrane receptors includes a decreased LH receptor mRNA as well as cellular internalization of the existing receptors. (Endocrinology 127: 2469-2474, 1990)
were the same, they would be inhibited by the intrauterine hormone milieu and unable to produce the high levels of testosterone needed for male genital differentiation (7, 8). The fetal-neonatal and adult populations of Leydig cells respond differently to high gonadotropic stimulation (3, 9-11). After human CG (hCG) stimulation, the adult testis shows a distinct LH receptor down-regulation and steroidogenic lesion (4-6), but both of these negative responses are missing in the fetal and neonatal testes. Instead, hCG stimulation increases the testicular LH receptor content and capacity to produce testosterone (3, 9-11). The molecular mechanisms responsible for these functional differences have so far remained unclear. As an attempt to delineate these mechanisms we now have studied the effects of hCG treatment on neonatal and adult testis mRNA levels of the LH receptor and of key steroidogenic enzymes. Besides elucidating these mechanisms in the adult testes, we also show that
2469
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 19:18 For personal use only. No other uses without permission. . All rights reserved.
hCG STIMULATION IN TESTIS
2470
clear-cut differences prevail between neonatal and adult rats in the expression of the LH receptor and specific steroidogenic enzyme genes.
Materials and Methods A single dose of hCG (Pregnyl, Organon, Oss, The Netherlands), 600 IU/kg body wt, was injected sc into neonatal (5day-old) and adult (60-day-old) male rats of the SpragueDawley strain produced in our own vivarium. The dose selected was based on earlier dose-response studies on hCG effects on rat testicular LH receptors and steroidogenesis (9). The rats were killed by decapitation 1, 2, or 3 days after hCG treatment. Trunk blood was collected and the serum separated and stored at —20 C. The testes were removed and either immediately snap-frozen in liquid nitrogen and stored at -70 C, or immersed directly into 4 M guanidinium thiocyanate reagent solution (GIT, Eastman Kodak Company, Rochester, NY; 0.5% sodium lauroylsarcosine, Sigma Chemical Company, St. Louis, MO; 25 mM sodium citrate, Sigma; 0.7% 2-mercapto-ethanol, Sigma; 0.1% antifoam A, Sigma) and stored, after homogenization, at -20 C. Serum testosterone and progesterone concentrations were measured from diethyl ether extracts by RIA as described previously (12, 13). The testosterone assay sensitivity was 1 fmol/tube; the intraassay coefficient of variation was less than 6%, and the interassay coefficient of variation less than 12%. The progesterone assay sensitivity was 4 fmol/tube; the intraassay coefficient of variation was 10%; and the interassay coefficient of variation 15%. The LH receptor measurements were performed as described before (9, 14, 15). A solid-phase lactoperoxidase method (16) was used for radioiodination of highly purified hCG (CR-121, 13,500 IU/mg by bioassay, prepared by Dr. R. E. Canfield, Columbia University, New York, NY, and provided by National Institute for Child Health and Human Development, Bethesda, MD). Total RNA was isolated from the deep-frozen testes by the proteinase K method (17) and from the GIT solution by a CsCl centrifugation method (18). The mRNAs were analyzed by a cDNA hybridization assay in slot blots (Minifold II, Scheicher & Schuell GmbH, Dassel, West Germany). Total denatured RNA (2-10 jug) was immobilized in each slot in GeneScreen hybridization membranes (Du Pont, NEN Research Products, Boston, MA). The RNA was fixed by UV irradiation for 5 min. Prehybridization and hybridization were performed at 42 C as described previously (19). The cDNA inserts were labeled by a Multiprime DNA labeling system (Amersham International pic, Amersham, Bucks, UK) with a [a-32P]deoxycytidine 5'triphosphate (3000 Ci/mmol). The cDNAs used were those of rat aromatase cytochrome P450 (P450arom) (20), human cholesterol side chain cleavage cytochrome P450 (P450scc) (21) and human 17-hydroxylase/17,20-lyase cytochrome P450 (P450cl7) (22). For LH receptor measurements a 40-mer oligonucleotide (nucleotides 232-271 of the rat LH receptor cDNA) (23) was synthesized and labeled by a DNA 3'-End Labeling Kit (Boehringer Mannheim GmbH, Mannheim, West Germany) with a [a-32P]dCTP (3000 Ci/mmol). The washed filters were autoradiographed using x-ray film
Endo • 1990 Vol 127 • No 5
(Kodak XAR-5, Eastman Kodak Company, Rochester, NY) and intensifying screens for 3-21 days at —50 C. Autoradiograms were scanned by an LKB UltroScan XL 2222-020 densitometer, and an LKB 2400 GelScan XL software package (LKB-Produkter AB, Bromma, Sweden) was used for the calculation of results. The mean control levels in each experiment were assigned values of 100%. Hybridization with a-actin cDNA demonstrated that similar amounts of RNA were present in parallel slots of the same experiments. Because expression of specific cDNA hybridization per a-actin hybridization did not affect the results, this correction was omitted in calculation of the final results. Each cDNA and oligonucleotide probe was hybridized with Northern blots of rat gonadal RNA, and hybridization only occurred with the RNA species of correct molecular sizes, according to literature (21-23). No hybridization was observed with RNA prepared for nonendocrine control tissue (liver). All samples to be compared were immobilized on the same membrane. The data were analyzed by one-way analysis of variance and Duncan's new multiple range test. When comparing two means the unpaired Student's t test was used. A P value less than 0.05 was chosen as the limit of statistical significance.
Results Injection of 600 IU/kg hCG increased serum testosterone levels at both ages studies, about 3-fold (P < 0.01) in 2 days in the neonates, and from 7-12-fold (P < 0.01) between 1-3 days in the adults (Fig. 1, upper panel). The other serum steroid analyzed was progesterone, which showed no change in the neonates (Fig. 1, lower panel). In contrast, in the adult, the basal concentration of progesterone increased as much as 12-15-fold on days 1 and 2 of the experiment (P < 0.01). The mRNA level of testicular P450scc increased between 1-3 days by 80-130% (P < 0.05) in the neonates and between 80-170% (P < 0.05-0.01) in the adults (Fig. 2, upper panel). The responses of the P450cl7 mRNAs were opposite at the two ages studied, displaying a greater than 4-fold increase (P < 0.01) in the neonatal testes but a 75% decrease (P < 0.01) in the adults (Fig. 2, middle panel). No response was observed in P450arom mRNA at either age (Fig. 2, lower panel). The unoccupied LH receptors and the respective mRNA levels before and after hCG treatment are shown in Fig. 3. In both parameters measured opposite effects were observed between the two ages. The number of unoccupied LH receptors was very low 1 day after hCG injection. It recovered rapidly by day 3 in the neonates but remained undetectable on days 2 and 3 in the adult testes. A 4-5-fold increase (P < 0.05) in LH receptor mRNA was observed in the neonates on days 2 and 3, and a concomitant decrease of 50% (P < 0.05) occurred in the adult testes at all 3 days after treatment. Table 1 presents the relative concentrations of the different parameters measured in the neonatal and adult
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 19:18 For personal use only. No other uses without permission. . All rights reserved.
hCG STIMULATION IN TESTIS
5.0
-1..
100 ADULT
0.0
0
1 2
3
I.I
40
0
••
7
60
20
300
**
80
III
% of Controi;
^ 10.0
2471
i
-111 0
1
2
200
100
0
NEONATE
.III 0
3
300
1
2
ADULT
NEONATE
1
2
3
150 NEONATE
.§
j> 0.3
a.
0
••
ADULT
15
0.4
2
100
3
600
20 0.5
200
Days After hCG Injection
Days After hCG Injection
|
ADULT
400
100
200
50
8
10
0.2
2 0.1 4>
0.0 0
1
2
3
0
1
2
3
3
Days After hCG Injection
200
High-dose stimulation with LH/hCG results in loss of LH receptors and blockade of the 17-hydroxylase/Cl720 lyase step in adult rat Leydig cells (4, 24). It is not yet known to what extent increased sequestration, internalization, and intracellular degradation, and decreased receptor protein synthesis are responsible for the LH receptor down-regulation. There is considerable information that the steroidogenic blockade is an estrogen mediated phenomenon, where intratesticular aromatization of androgens and estrogen receptor mediated synthesis of a specific protein in Leydig cells play a role (24, 25). Previous studies have shown increased receptor internalization and degradation during down-regulation in
200 NEONATE
so
Discussion
1
Days After hCG Injection
FlG. 1. Serum testosterone (upper panel) and progesterone (lower panel) in the serum of neonatal (5-day-old, left) and adult (60-day-old, right) male rats before (day 0) and 1, 2, and 3 days after a 600 IU/kg body wt sc injection of hCG. Each bar is the mean ± SE of measurements from 5-12 animals. The asterisks indicate significant differences from the 0-day level (**, P < 0.01, Duncan's test).
rat testis tissue or serum. The basal serum concentrations of progesterone and testosterone were both 2-fold higher in the adult rat. No difference occurred between testicular concentrations of the cholesterol side chain cleavage and aromatase mRNA levels, but that of 17hydroxylase/17,20-lyase was 2.7-fold higher in the adult testis. The LH receptor mRNA concentration was 10fold higher in the adult testis.
0
100
ADULT
Illl llll 0
1
2
3 0 Days After hCG Injection
1
2
3
FIG. 2. Testicular concentrations of mRNAs for P450scc (upper panel), P450cl7 (middle panel) and P450 arom (lower panel) before and up to 3 days after a sc injection of 600 IU/kg hCG. ADU, arbitrary densitometric unit. Each bar is the mean ± SE of measurements from 5-12 animals. *, P < 0.05; **, P < 0.01 as compared with 0-day level.
adult testes (4). Our present data suggest that the rate of receptor synthesis also may be reduced, although they do not provide direct evidence whether the suppressed steady state level of LH receptor mRNA is due to decreased synthesis or increased degradation of the message. The loss of mRNA was less pronounced than that of the receptor protein. This is logical, because the actual receptor loss is the sum of several negative effects, i.e. increased receptor occupancy, increased receptor sequestration and intracellular degradation, decreased LH receptor gene transcription or increased mRNA degradation, and decreased protein synthesis. The LH receptor mRNA levels in fact only monitor the last responses. A decrease of receptor mRNA levels has been demonstrated
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 19:18 For personal use only. No other uses without permission. . All rights reserved.
hCG STIMULATION IN TESTIS
2472 1.5
0.15 NEONATE
0.10
1.0
0.05
0.5
0.00
0
1 2
ADULT
0.0
3
0
1 2
3
1 2
3
Days After hCG Injection
800 ADULT
NEONATE
600 -
100
400 50 200
0
1
2
3
0
Days After hCG Injection
FIG. 3. Testicular concentrations of LH receptors (upper panel) and the mRNA for LH receptor (lower panel) before and up to 3 days after a 600 IU/kg sc injection of hCG. ADU, arbitrary densitometric unit. Each bar is the mean ± SE of measurements from 5-12 animals. *, P < 0.05; **, P < 0.01 as compared with 0-day level. TABLE 1. Comparison between the neonatal and adult male rats of relative abundances of serum (S) progesterone and testosterone, and on testicular mRNAs of P450scc, P450cl7, P450arom, and LH receptor
S-progesterone S-testosterone P450scc P450cl7 P450arom LH receptor
Neonates
(n)
Adults
(n)
P value
100 ± 17 100 ± 27 100 ± 30 100 ± 27 100 ± 6 100 ± 36
(10) (7) (5) (5) (15) (10)
218 ± 64 209 ± 20 83 ±20 267 ± 33 80 ± 7 1075 ± 167
(5) (5) (5) (5) (14) (10)
