The role of LH in regulation of leydig cell responsiveness to an LHRH agonist

131

Molecular and Cellular Endocrinology, 33 (1983) 131-146 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01068

THE ROLE OF LH IN REGULATION OF LEYDIG CELL RESPONSIVENESS TO AN LHRH AGONIST Richard M. SHARPE MRC, Reproductioe Biology Edinburgh EH3 9E W (U.K.)

and Ham&h M. FRASER Unit, Centre for Reproductive

BioIogy, 37 Chalmers

Street,

Received 31 May 1983; accepted 29 July 1983

The short-term relationship between deprivation of LH and changes in the responsiveness of isolated Leydig cells to an LHRH agonist was studied in adult rats injected with a potent antiserum to oLH. This treatment suppressed serum and intra-testicular levels of testosterone within 4 h of injection by SO-904 and 708, respectively, with further suppression to levels found in hypophysectomized rats by 20 h. At 12-20 h after antiserum injection there was no major consistent change from control values in the number of Leydig cell LH (hCG)-receptors or in the testosterone response of the cells to hCG-stimulation. In the same cells the number of LHRH-receptors and the maxima1 testosterone response to LHRH agonist were increased (Pi 0.001) by 50% or more, although the magnitude of increase varied considerably between experiments. The ability of LHRH agonist to enhance the testosterone response of cells to hCG-stimulation was also increased significantly (P < 0.001) in antiserum-treated rats. Comparable changes in testosterone responsiveness to LHRH agonist were observed following purification of Leydig cells on Percoll gradients. Although there was some evidence for changes in Leydig ceil LHRH-receptor numbers and responsiveness to LHRH agonist at 4 h after intravenous antiserum injection, these changes were small compared with changes seen at later times. it is concluded that Leydig cell responsiveness to an LHRH agonist is negatively regulated by LH, and this has implications with respect to a possible role of ‘testicular LHRH” in regulation of the intra-testicuIar levels of testosterone. Iceywords: antiserum to oLH; LH-receptors; intra-testicular testosterone.

LHRH-receptors;

serum testosterone;

In recent years it has been established that luteinizing hormone-releasing hormone (LHRH) and its ago&tic analogues can exert major direct zffects on testicular steroidogenesis by interacting with specific LHRHreceptors located on the Leydig cell surface (Hsueh and Jones, 1981; Sharpe, 1982; Sharpe and Harmar, 1983). These actions are believed to reflect those of an endogenous ligand, ‘testicular LHRH’, secreted by the Sertoli cells (Sharp et al., 1981, 198213; Sharpe and Harmar, 1983; 0303-7207/83/$03.~

0 1983 Elsevier Scientific Publishers Ireland, Ltd.

132

R.M. Sharp

and H.M. Fraser

Nagendranath et al., 1983; Bhasin et al., 1983). Although the effects of LHRH on the Leydig cell were considered originally to be exclusively inhibitory, it is now clear that LHRH agonists can also stimulate testosterone secretion, as this has been demonstrated in vitro (Hunter et al., 1982; Sharpe and Cooper, 1982a, b) and in vivo in both hypophysectomized (Sharpe et al., 1982a; 1983b) and intact (Sharpe et al., 1983a, b) rats. From the latter studies in vivo it appears that a crucial factor in determining whether LHRH exerts a stimulator-y or an inhibitory effect on Leydig cell steroidogenesis is the degree of concomitant exposure to LH or hCG (see Sharpe et al., 1983b), stimulatory effects only being evident in the presence of normal or subnormal levels of gonadotropin. These observations fit well with changes in the number of Leydig cell LHRH-receptors, which have been reported to increase after hypophysectomy (Bourne et al., 1982; Sharpe et al., 1982a) and to deerease after exposure to supraphysiolo~cal levels of gonadotrophin (Sharpe, 1982; Sharpe and Rommerts, 1983). Such findings could be interpreted as evidence that the responsiveness of Leydig cells to LHRH and to ‘testicular LHRH’ may be determined by the level of exposure to LH and/or by the intra-testicular levels of testosterone. The present study was designed to extend the above observations by monitoring changes in Leydig cell LHRH-receptors numbers and responsiveness to an LHRH agonist in vitro following short-term (4-24 h) suppression of Inca-testicular levels of testosterone by neutralizing circulating LH using an antiserum to ovine LH.

MATERIALS

AND

METHODS

Animals and treatments The animals used were Sprague-Dawley rats (aged between 6.5 and 85 days) from our own colony and housed under conventional conditions. Five experiments were performed to investigate the relationship between the period of suppression of LH and functional changes in the Leydig cells. In Expts. l-3, groups of 4 rats of the same age were injected subcutaneously with either 1 ml vehicle (0.9% saline in Expts. 1 and 2, normal rabbit serum in Expt. 3) or with 1 ml rabbit antiserum to oLH (see below) and killed with dry-ice-generated CO, at specified intervals after injection when isolated Leydig cells were prepared as described below. In Expts. 4-5, groups of rats were lightly anaesthetized with ether and injected intravenously via the tail vein with either 0.5 ml 0.9% saline or with 0.5 ml antiserum to oLH. Groups of 4 control and 4 antiserumtreated rats were then killed at either 4 or 20 h after injection and

Leydig cell responsiveness

to LHRH

133

isolated Leydig cells prepared as described below. For investigation of antiserum effects at 12 and 20 h after treatment, injections were timed so that animals were killed between 0800 and 0900 h, whilst for investigation of the effects of antiserum exposure for 4 h, rats were injected at 0800 h and killed at 1200 h; control rats were treated similarly. To test the biological effectiveness of the injected antiserum to oLH, two further experiments were performed (Expts. 6 and 7). In Expt. 6, groups of rats were injected subcutaneously with either 1 ml saline or 1 ml antiserum as detailed above and, at 4 and 20 h after injection, groups of 4 rats from each treatment group were killed with dry-ice-generated CO,, blood collected immediately by decapitation, and the paired testes removed and testicular interstitial fluid collected as described previously (Sharpe and Cooper, 1983). Similar procedures were followed in Expt. 7, except that the rats were injected intravenously under ether anaesthesia with either 0.5 ml saline or 0.5 ml antiserum. Preparation‘ of dispersed Leydig cells

Dispersed testicular cells were prepared essentially as described previously (Sharpe and Cooper, 1982a, b). Briefly, groups of 4 decapsulated testes were placed into 50 ml conical glass flasks together with 1.5 ml/testis of Krebs-Ringer bicarbonate solution (KRB) containing 0.25 mg/ml collagenase (Type 1; Sigma) and 2.5 mg/ml bovine serum albumin (BSA; fraction V, Sigma), and dispersed by incubating for 8 min at 34’C in a shaking water bath (170 cycles/mm). The dispersed tissue was then diluted to 50 ml with KRB and tubular tissues allowed to settle. The cells in the supernatant were then precipitated by gentle centrifugation (250 X g for 5 min) at 4’C and resuspended at high concentration (8-10 X lo6 nucleated cells/ml) in medium 199 (Flow Laboratories, U.K.) containing Hanks’ salts, 20 mM Hepes and 2.5 mg/ml BSA (= medium M199H). The nucleated cell concentration was then determined using a haemocytometer, and a small aliquot of cells removed, diluted to 3 X lo6 cells/ml and air-dried cell smears prepared on glass slides. When dried, the smears were fixed for 10 min in acetone and stained for 3/?-hydroxysteroid dehydrogenase (3P-HSD) as detailed below. The remaining isolated cells were used for measurement of testosterone production and in some instances for measurement of LHand LHRH-receptors as detailed below. In Expt. 3 a proportion of the suspension of testicular cells from control and antiserum-treated animals was purified on a discontinuous 50 ml gradient of Percoll (Pharmacia) in M199. The gradients were comprised of successive 10 ml layers of Percoll with average densities of 1.09, 1.07, 1.05 and 1.03 g, topped with a layer of 10 ml medium alone.

134

R.M.

Shorpe and H.M.

Fraser

Appro~mately 50 X lo6 nucleated cells were layered on to each gradient and centrifuged at 4*C for 20 min at 800 X g. Cells contained in the layer of Percoll of average density 1.07 were then aspirated, washed and precipitated by centrifugation at 250 x g for 5 min. The precipitated cells were then resuspended in 2 ml medium, nucleated cells counted in a haemocytometer and cells used for assessment of testosterone production as described below. In routine practice, between 65 and 80% of the cells purified in this manner are identifiable histochemically as Leydig cells (see below).

Cells staining positively for 3/3-HSD were taken to represent Leydig cells, and the percentage of such cells was determined using air-dried cell smears rather than isolated cell suspensions as used previously (e.g. Sharpe and Cooper, 1982a, b), because recent evidence suggests that 3,&HSD reactions using intact cell suspensions may identify cells other than Leydig cells (e.g. see Aldred and Cooke, 1983; Molenaar et al., 1983). To stain the fixed cells for 3/&HSD, slides were incubated at room temperature for 2 h in a solution which comprised in final form: 0.1 M Tris-HC1(13%), N, N-dimethyl formamide (5.4%; Sigma), sodium cyanide (0.0003%) and anhydrous magnesium chloride (0.0004~) in distilled water, and in which were dissolved the following reagents and their final concentrations: nitro-blue tetrazolium (0.82 mg/ml; Sigma) dehydroepiandrosterone (0.27 mg/ml; Sigma) and ~-nicotinamide adenine dinucleotide (0.91 mg/ml; Sigma), Full details of the preparation of the stock solutions and the methods used can be found elsewhere (Lojda et al., 1979). Following incubation, slides were washed with water, mounted in glycerine jelly and the percentage of stained nucleated cells determined by counting at least 4 random fields containing 250 or more cells using a graticule. The percentage of 3&HSD-positive cells in the various experiments did not differ significantly between control and antiserum-treated animals, although there was a trend towards higher nucleated cell yields associated with a lower percentage of 3~-HSD-positive cells in rats treated 20 h previously with the antiserum (W 3~-HSD-positive cells at 4 h after injection = 19.9 k 6.4 (control) and 18.0 + 5.0 (anti-oLH), mean + range, N = 2; at 20 h = 24.7 f 11 (control), 17.8 4 8.0 (anti-oLH), mean + range, N = 5). Assessment of testosterone production in vitro For measurement of testosterone production,

concentrated

cell sus-

Leydig cell responsiveness to LHRH

135

pensions were diluted to 0.5 x lo6 nucleated cells per ml using medium 199 to which was added Earle’s salts, sodium bicarbonate (0.22%), rglutamine (2 mM; Flow Laboratories), transferrin (5 pg/ml; Sigma) insulin (10 mg/ml; Sigma), ceruloplasmin (1 U/ml; Sigma), penicillin (100 IU/ml; Flow Laboratories), streptomycin (100 pg/ml; Flow Laboratories), fungizone (2.5 pg/ml; Flow Laboratories) and BSA (2.5 mg/ml) (= medium M199E). After addition of hormones at the required concentration, aliquots of 0.275 X lo6 cells were incubated in a final volume of 0.65 ml in plastic multiwell dishes (Nunc, Denmark) for 5 h at 34°C under a humidified atmosphere of 5% CO*: 95% air. At the end of incubation the medium was aspirated, centrifuged for 5 min at 1000 X g and the supernatant stored at -20°C prior to measurement of testosterone. Identical procedures were used for incubation of Percollpurified cells (Expt. 3), except that the number of nucleated cells added per well was reduced to 0.05 X 106. The hormones added to cell incubations were either hCG (Chorulon, Intervet) or an LHRH agonist ((D-Sert-bu6, des-Gly-NH:‘) LHRH ethylamide; Hoechst). Measurement of LH- and LHRH-receptor numbers Measurement of the binding of [ 12’I]hCG and [ lZf]LHRH agonist to Leydig cell LH (hCG)- and LHRH-receptors, respectively, was performed essentially as described previously (Sharpe and Cooper, 1982b). For both receptor assays, M199H was used throughout and the number of nucleated cells added per tube varied between experiments from 0.9-2.84 x lo6 for LHRH-receptor measurement and from 0.7-2.6 X lo6 for hCG-receptor measurement, although within any single experiment equal numbers of cells from control and antiserum-treated rats were always used. Assays were performed in the presence of saturating concentrations of the respective radioligands (100 ng/ml for [‘251]hCG; 400 x lo3 cpm for [‘251]LHRH agonist) and non-specific binding was assessed by addition of a lOOO-fold excess of unlabelled hormone; all incuba~ons were run in replicates of 4-6. Receptor assays were performed at 21T for either 45 min (LHRH) or 18 h (hCG) and termination of incubation and the separation of free and bound hormone were performed as described previously (Sharpe and Cooper, 1982b). Radioiodi~ation of hormones hCG (NIAMDD-hug CR119), LHRH agonist, rat LH (NIADDK rLH-I-6) and rat FSH (NIADDK-rFSH-I-5) were iodinated using lactoperoxidase (Miyachi et al., 1972) to specific activities of approximately 50, 1000, 100 and 120 mCi/mg, respectively.

136

R. M. Sharpe and H. M. Fraser

Measurement of testosterone Testosterone was measured by radioimmunoassay (Corker and Davidson, 1978), as described previously (Sharpe and Cooper, 1983). Serum was extracted with hexane : diethyl ether prior to assay, whilst incubation medium and testicular interstitial fluid were assayed direct after appropriate dilution.

Production of LM antiserum Each of 3 adult male New Zealand White rabbits was immunized with 20 pg oLH (NIA~DD-oLH-S23) dissolved in 0.9% saline and emulsified with Freund’s complete adjuvant. Each rabbit was injected with a total of 2 ml emulsion dist~but~d between 15 intradermal sites. Rooster immu~zations were given 12, 20 and 34 weeks later, samples of blood collected between 10 and 20 days after each immunization and the separated serum stored at -40°C. All rabbits produced high titre antibodies, and antiserum obtained from one animal (R-31) after the first booster immunization was used in the present study. Titration curves prepared by incubating dilutions of the antiserum under standard radioimmunoassay conditions showed that the titres of the antiserum, expressed as the final dilution binding 33% of [*251]oLH or [lz51]rLH, were 1 : 307200 and 1 : 153 600, respectively. The antiserum also bound [‘251]rFSH but at a much lower titre (1: 900), and it is unlikely that this would have significant effects on the biological actions of FSH in antiserum-treated animals.

Statistical analysis Results were analysed using 2-factor analysis tion) and Student’s t-test.

of variance

(with replica-

RESULTS

Biological effectiveness of the antiserum to oLH The biological effectiveness of the LH antiserum was clearly evident from the reduction in testicular and serum levels of testosterone that occurred following its subcutaneous or intravenous a~istration (Fig. 1). Thus, serum levels of testosterone were reduced by gO-90% within 4 h of antise~m injection and had declined to castrate levels (0.3 ng/ml) by 20 h, irrespective of the route of a~nistration. Although levels of testosterone in testicular interstitial fluid also declined rapidly, the reduction in testosterone levels was somewhat slower than that observed for serum testosterone with only a 70% fall in testosterone levels by 4 h but a reduction to levels found in hypophysectomized rats (Sharpe et al., 1982a) by 20 h (Fig. 1).

137

Leydig cell responsiveness to LHRH

PERIPHERAL LEVELS TESTOSTERONE

OF

INTRA-TESTICULAR OF

INTRAVENblS

LEVELS

TESTOSTERONE

ROUTE I ,700

600 500

;;I 2 0

400

= z

300

5 ;;I

200

,% **

100

I

0

: 2 -_( s r ? 5 0 ;;t

SUBCUTANEOUS

2

ROUTE 500

: ;;I

400

;

300

q 0

200

5 ;

2tLYL TCrz

3-

1

0

31% *+

21% **

+4h

100

10% ***

+

TIME

20h

AFTER

+4h

0

INJECTION

Fig. 1. Biological effectiveness of the antiserum to oLH as judged by changes in the serum and ~tra-testicul~ levels of testosterone. Data for spine-injected rats is shown in the open columns and that for antiserum-injected rats in the closed columns; the mean values for the latter groups are also expressed as a percentage of the respective mean control value, Injections were given either intravenously (0.5 ml; top) or subcutaneously (1.0 ml; bottom) to 69-(top) or 82-(bottom) day-old rats, and the illustrated values are the mean & SD for 4 rats at each time-point. ** P < 0.01, *** P r: 0.001, compared with respective control group.

Effect of antiserum to oLH on Leydig cell LH- and LHRH-receptor numbers At 4 h after subcutaneous injection of 1 ml antiserum, there was a small but statistically significant increase in the number of LH (hCG)-receptors per Leydig cell (Fig. 2), although this increase was not evident at 4 h after intravenous injection of the antiserum (controls, 4.87 rfr0.2 ng

T-production

(ng): * f f

1** 3 2***

1.2 ***

0.1

4 8 5

lo* 1* 144_cll 641 2**

16k 158+ 44*

_

_

Expt. 2

cells

responsiveness

per lo6 3/3-HSD-positive

and in testosterone

0.1 1.7

12 157 57

* 1 ** *11 2*** *

12.0* 1.7 **

0.2

* 1 & 7 * 1

5.3*

17 135 38

4.8* 8.1&

Expt. 3

of Leydig cell isolated

* 2 + 10 I 4

0.6

9 1x9 71

* 1** +12** 5 *** *

22.2 & 0.7 ***

_

12 142 31

13.0+

_

Expt. 4

rats

in the

from the testes of control

Injections were given either subcutaneously (Expts. l-3) or intravenously (Expt. 4). Data for testosterone responsiveness is for cells incubated presence of maximally stimulating concentrations of hCG (5 nM) or LHRH agonist (lo-’ M). Values are the mean+ SD of triplicate (T-responsiveness) or quadruplicate (receptor measurements) incubations of cells. * P < 0.05, ** P < 0.01, *** P < 0.001 , in comparison with respective value for cells from control rats.

Basal hCG-stimulated LHRH-A stimulated

12 102 60

4.3+

receptors

38.3 *

LHRH-A

0.1 2.0

* 1 +13 f 1

(cpm x 10 ‘)

15 103 31

11.k

4.1*

Expt. 1

Leydig cells from rats treated with anti-oLH 20 h previously: hCG(LH)-receptors (ng)

Basal hCG-stimulated LHRH-A stimulated

rats:

(cpmX 10e3)

(ng):

receptors

T-production

LHRH-A

Leydig cells from control hCG(LH)-receptors (ng)

numbers to oLH

Values expressed

Summary of changes in LH- and LHRH-receptor and rats treated 20 h previously with antiserum

Table 1

z %

E 01 6 3 fi

139 LH-RECEPTOR

NUMBERS

67

LHRH-RECEPTOR

40-l

NUMBERS

~

CONTROL TIME OF

AFTER ANTISERUM

INJECTION TO

oLH

Fig. 2. Changes in the number of LH- and LHRH-receptors in Leydig cells from control rats and rats injected subcutaneously 4, 12 or 20 h previously with 1 ml antiserum to oLH (Expt. 1). Values are the mean f SD for 4 (antiserum-treated) or 8 (control) incubations, with data for controls deriving from two separate cell preparations which did not differ significantly from each other. ** P < 0.01, *** P < 0.001, compared with control group.

hCG bound/l@ 3~-HOD-positive cells; anti~~m-treated, 4.64 & 0.3 ng; mean & SD, n = 4). At 12 and 20 h after subcutaneous injection of antiserum no significant change in the number of LH-receptor was observed (Fig. 2, Table 1). In contrast, in the same cells the number of LHRH-receptors showed dramatic changes. Thus at 4 h after subcutaneous injection of antiserum there was a small but statistically insignificant increase in LHRH-receptors (Fig. 2), a finding duplicated after

R.M. Shurpe and H.M. Fraser

140

TESTOSTERONE -INCUBATED

ONSE

FlE$

TO

5nM

hCG

INCUBATED

WITHOUT

WITH

LHRH-A

220

180

140

100. 3 Ii w ._

60

1

TESTOSTERONE

RESPONSE

TO

IDM

hCG

e INCUBATED

Y?

WITHOUT

!

INCUBATED

-BASAL

TESTOSTE

INCUBATED

ONE

WITHOUT

20

nliii ONTROL

TIME OF

+4h

*** 11 WITH

LHRH-A

30

0

et*

PRODUCTION

INCUBATED

LHRH-A

10

WITH

LHRH-A

LHRH-A

+12h

AFTER ANTISERUM

t20h

INJECTION TO

oLH

L

+4h

***

+12h

TIME

AFTER

OF

ANTISERUM

+20h

INJECTION TO

oLH

Fig. 3. Changes in basal, LHRH agonist- and hCG-stimulated testosterone production by Leydig cells from control rats and rats injected subcutaneously 4,12 or 20 h previously with 1 ml antiserum to oLH (Expt. 1). Incubations were performed without addition of hCG (bottom panel) or in the presence of a submaximally stimulating (middle panel) or maximally stimulating (top panel) concentration of hCG, and each set of incubations was run in the absence (left) or presence (right) of lo-’ M LHRH agonist. Values are the mean+ SD for either 3 (~tise~-treated) or 6 (control) incubations, and other details are as given in the legend to Fig. 2.

Leydig cell responsiveness to LHRH

141

injection of the antiserum intravenously (controls, 9.1 f 1.0 X lo3 cpm [12’I]LHRH agonist bound/lo6 3/3-HSD-positive cells; antiserum-treated, 10.9 k 0.6 x lo3 cpm; mean + SD, n = 4) and which attained statistical significance (P < 0.05). By 12 and 20 h after antiserum injection there was a substantial (P < 0.001) increase in LHRH-receptor numbers (Fig. 2) although it should be noted that the magnitude of this increase varied considerably between experiments (Table 1). Effect of antiserum to oLH on testosterone responsiveness of isolated Leydig cells Testosterone responsiveness of isolated cells was studied under three conditions: (1) in the complete absence of hCG (= basal production); (2) in the presence of a concentration of hCG (1 PM) which elicited approximately 20-30% of the maximum response; and (3) in the presence of a concentration of hCG (5 nM) in excess of that required to elicit a maximum testosterone response (e.g. see Sharpe and Cooper, 1982a). Cells from control and antiserum-injected animals were incubated under these conditions either in the absence or the presence of a maximally stimulating concentration (10m7 M) of LHRH agonist (Sharpe and Cooper, 1982a). Except at 4 h after subcutaneous injection, the basal production of testosterone by isolated cells from antiserum-injected rats was always significantly lower than the corresponding values for control animals (Figs. 3 and 4, Table 1). In Expts. 1 and 2, the testosterone response of cells from control and antiserum-treated rats to either submaximal or maximal hCG-stimulation did not differ significantly, although there were differences in Expts. 3 and 4. In the former, the response of cells from antiserum-injected animals to a submaximally stimulating concentration of hCG was increased significantly compared with controls, a a change that was still evident after Percoll purification of the Leydig cells (Fig. 4), whilst in Expt. 4 the maximal response of cells from antiserum-treated rats to hCG was increased compared with controls (Table 1). It is unknown whether these changes in response to hCG are a consequence of LH-deprivation or whether they reflect errors in Leydig cell identification. The testosterone response of isolated cells to LHRH agonist alone was not increased at 4 h after subcutaneous injection of antiserum (Fig. 3) but was increased slightly (P < 0.05) at 4 h when antiserum was administered intravenously (control, 40 f: 1 ng/106 3P-HSD-positive cells; antiserumtreated, 50 + 4 ng; mean f SD n = 3), there being no significant change in the maximal response of the same cells to hCG (control, 175 + 10 ng; antiserum-treated, 155 + 15 ng; mean + SD, n = 3). At 12 and 20 h after

142

R. M. Sharpe und H. M. Fraser NON j 2004

PURIFICD

INCUBATED WITHOUT LHRH-A

j

CELLS INCUBATED LHRH

PERCOLL-PURIFIED

WI?,, A

CELLS

x ID

450

0

e

0”

150-

INCUBATED WITHOUT LHRH-A

***

INCUBATED LHRH-A

WITH

,1A I**

t; ,”

BASAL

hCG

hCG

+~DM hCG

Fig. 4. Changes in basal, LHRH agonist- and hCG-stimulated testosterone production by Leydig cells from control rats (open columns) and rats injected subcutaneously 20 h previously with 1 ml antiserum to oLH (solid columns; Expt. 3). Incubations were performed as detailed in the legend to Fig. 3, althou~ the layout of the figure is different. Data are shown for non-purified cells (top) and for the same preparations of cells after purification on Percoll density gradients (bottom). Values are the mean + SD for triplicate incubations. ** P i 0.01, ***P < 0.001, compared with respective control column.

injection, the testosterone response to LHRH agonist of cells from antiserum-injected animals was always increased considerably when compared with control (Figs. 3 and 4, Table l), although the magnitude of this increase varied considerably and bore some relationship to the magnitude of increase in LHRH-receptors numbers (Table 1).

When cells from control rats were incubated in the presence of hCG and LHRH agonist, the latter enhanced the testosterone response of these cells to hCG (e.g. Fig. 3) although this interactive effect was only evident in 4 of 6 experiments; similar .findings have been reported previously (Sharpe and Cooper, 1982a). This interactive effect of LHRH agonist and hCG was much more pronounced in rats treated with antiserum to oLH. Thus at 12-20 h after injection of the latter, LHRH agonist significantly enhanced the testosterone response of the cells to hCG and this enhancement was always of greater magnitude (P < 0.01 to P < 0.001) than that observed with cells from control rats (e.g. Figs. 3 and 4, but not all data shown) and was observed in every experiment. The increase in testosterone responsiveness to LHRH agonist of cells from antise~m-tr~ted rats detailed above, was equally evident in nonpurified ( - 20% Leydig cells) and Percoll-purified ( - 70% Leydig cells) cells (Fig. 4), suggesting that the observed changes were not artifacts related to misidentificatidn of cells or to alteration in the proportion of damaged cells. This is e,mphasized further by comparison of the maximal responsiveness of individual cell preparations to LHRH agonist on the one hand, and to hCG on the other. In the four experiments detailed in Table 1, the mean (4 SD) response of cells from control rats to LHRH agonist alone was 27 + 3% of the response of the same cells to maximally stimulating concentration of hCG, whereas the same comparison for cells from antise~m-trite rats yielded a value of 44 + 99, a difference that was statistically si~fic~t (P < 0.01) Moreover, this selective increase in Leydig cell responsiveness to LHRH agonist was equally evident in Expt. 4, in which the response of cells from antiserum-treated rats to hCG was increased, and in the other experiments (l-3) in which there was no change in hCG-responsiveness (Table 1).

DISCUSSION The present findings demonstrate that deprivation of intact male rats of Lo-stimulation, and the resulting drop in Inca-testicul~ levels of testosterone, are associated with a rapid and pronounced increase in the number of Leydig cell LHRH-receptors and in the testosterone responsiveness of these cells to an LHRH agonist. These changes suggest that there may be an inverse relationship between the level of exposure to endogenous LH and Leydig cell responsiveness to LHRH agonist. It is also not unreasonable to expect that Leydig cell responsiveness to endogenous ‘testicular LHRH’ is increased similarly. However, before

144

R.M. Shurpe and H.M. Fruser

considering the possible imp~cations of these findings, it is appropriate to consider first the validity of the present observations. Because results in the present study are expressed in units per Leydig cell, it is obvious that misidentification or underestimation of the number of such cells could lead to artifactual changes in the number of Leydig cell LHRH-receptors and in the responsiveness of the cells to LHRH agonist. The fact that the percentage of cells identified as Leydig cells (i.e. 3fi-HSD-positive cells) was consistently, although not significantly, lower for ceils obtained from the testes of antiserum-treated rats than for cells from control rats, underlines this possibility. However, there are several good reasons for considering that this interpretation is unlikely. Perhaps the most important of these is that in the same cell preparations in which changes in LHRH-receptors and LHRH-responsiveness were observed, no consistent change was noted in the number of LH (hCG)receptors or in responsiveness to hCG. This was endorsed further by the observation that in 4 separate experiments the testosterone response to LHRH agonist, when expressed as a percentage of the maximal response to hCG, was 44 + 9% for cells from antiserum-treated rats compared with 27 f 3% for cells from control rats. This observation also negates the possibility that Leydig cells from ~tiserum-treated rats were in some way less susceptible to damage induced by the isolation procedure, as in these circumstances comparable changes in the response of the cells to hCG and LHRH agonist would have been anticipated. Again, even after purification of the crude Leydig cell suspension on Percoll gradients, a procedure which has been shown to increase both the purity and ‘quality’ of the resultant Leydig cells (see Aldred and Cooke, 1983), differences between cells from control and antiserum-treated rats in their response to LHRH agonist were still evident. The present findings are also consistent with two previous studies using hypophysectomized rats (Bourne et al., 1982; Sharpe et al., 1982a), both of which demonstrated a doubling in the number of LHRH-receptors per Leydig cell. In the first of these reports this change was reported to occur as early as 24 h after hypophysectomy, an observation which ties in closely with the present demonstration of an increase in Leydig cell LHRH-receptor numbers at 12 h after antiserum injection, with evidence for some change as early as 4 h when the antiserum was administered intravenously. In the present experiments, as in previous studies in hypophysectomized rats (Sharpe et al., 1982a), there appeared to be a degree of correlation between the magnitude of the antiserum-induced increase in LHRH-receptor numbers and the increase in testosterone responsiveness to LHRH agonist. This may imply that it is the change in number of LHRH-receptors which is of primary importance, and it is therefore of

Leydig cell responsiveness

to LHR H

145

interest to identify what triggers this change in antiserum-treated rats. There are two distinct possibilities: first, that it is a consequence of the reduction in intra-testicular levels of testosterone or, second, that it results from the reduction in stimulation of the Leydig cell by LH. It is important to distinguish between these possibilities as the second option would imply an intrinsic function of the Leydig cell whereas the first possibility could involve the Sertoli cells, as these are an important site of action of testosterone within the testis. Experiments involving injection of testosterone at the time of antiserum administration might enable this problem to be resolved, and experiments along these lines are in progress. From studies on the testis in vitro and in vivo, it now seems clear that LHRH and its agonists can interact locally with LH to modulate intra-testicular testosterone levels and secretion (Sharpe et al., 1983a, b). In the light of such findings it has been suggested that the function of ‘testicular LHRH’ might be to ensure that intra-testicular levels of testosterone do not fall below the optimum required for the support of spermatogenesis (Sharpe and Rommerts, 1983), a mechanism that might be important in view of the episodic nature of LH release (see also Sharpe and Cooper, 1983). Such a mechanism would have to be geared to the degree of exposure to LH, and it is in this context that the present findings are of significance as they demonstrate that Leydig cell responsiveness to an LHRH agonist increases rapidly following LH deprivation. As the opposite change has been shown to occur after hCG injection (Sharpe and Fraser, 1983; Sharpe ‘and Rommerts, 19X3), the available findings are at least consistent with the role postulated for ‘ testicular LHRH’, although proof of the existence of such a mechanism must await detailed studies in vivo.

ACKNOWLEDGEMENTS We are grateful to Irene Cooper and Mariwen Swaney for skilled help, to Dr. Jurgen Sandow (Hoechst) for the gift of LHRH agonist, to Rachel Popkin for iodinated rat LH and to the NIAMDD, U.S.A., for hCG for radioiodination.

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