Intratesticular regulation of testosterone secretion: comparison of the effects and interactions of hCG, an LHRH agonist and testicular interstitial fluid on Leydig cell testosterone secretion in vitro

241

Molecular and Cellular Endocrinology, 41 (1985) 241-255 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01342

Intratesticular regulation of testosterone secretion: comparison of the effects and interactions of hCG, an LHRH agonist and testicular interstitial fluid on Leydig cell testosterone secretion in vitro Richard M. Sharpe MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland (U.K.) (Received

Keywords:

Leydig control

cell; testicular mechanisms.

interstitial

21 January

fluid;

LHRH

1985; accepted

agonist;

LHRH

15 April 1985)

antagonist;

hCG;

testosterone

secretion;

intratesticular

Summary

The stimulatory effects on Leydig cell testosterone secretion of a polypeptide(s) factor, present in testicular interstitial fluid (IF) were compared with those of hCG and an LHRH agonist (LHRH-A). The actions of IF and LHRH-A were similar in showing (1) a delayed onset of action, (2) enhancement of testosterone production in response to a maximally stimulating concentration (5 nM) of hCG, and (3) near cessation of stimulation following their removal from the incubation medium. However, addition of an LHRH antagonist blocked only the actions of LHRH-A. Moreover, IF continued to stimulate testosterone production up to at least 20 h either on its own or in the presence of 5 nM hCG, whereas the stimulatory effects of LHRH-A disappeared beyond 6 h. IF was also able to enhance testosterone production in response to LHRH-A or in response to hCG + LHRH-A. IF enhanced testosterone production over 4-20 h in response to all doses of hCG and increasing concentrations of IF caused dose-dependent increments in the rate of hCG (5 nM) -stimulated testosterone production. With submaximally stimulating doses of hCG or with LHRH-A alone, the stimulatory effect of IF was more or less additive, whereas with maximally stimulating doses of hCG the effect of IF was clearly synergistic. Thus, whereas the rule of testosterone production by Leydig cells in response to 5 nM hCG declined progressively from 4 to 20 h, addition of IF attenuated or prevented this decline. These findings have implications with respect to the physiological control of intratesticular testosterone levels and with respect to the regulation of steroidogenesis. They also imply that isolation of Leydig cells from their normal stimulatory environment results in a decrease in their steroidogenic responsiveness.

Because of the dependence of mammalian spermatogenesis on high intratesticular levels of testosterone, a strong theoretical case can be made for a need for local mechanisms capable of controlling testosterone levels within the testis (see Sharpe, 1983, 1984, for reviews). It is presumed that such mechanisms would involve the production, probably by the Sertoli cells, of factors which 0303-7207/85/$03.30

0 1985 Elsevier Scientific

Publishers

Ireland,

acted on the Leydig cells either to control testosterone production directly or, more likely, to modulate testosterone production in response to pituitary luteinizing hormone (LH). There is considerable morphological evidence for Sertoli cell regulation of the Leydig cells (e.g. Kerr et al., 1979, de Kretser, 1982; Bergh, 1983), and as a result of such interactions there can be marked Ltd.

248

changes in the steroidogenic responsiveness of the Leydig cells to LH or hCG (e.g. Risbridger et al., 1981; Sharpe et al., 1984). However, the agents involved in these interactions remain unidentified. One factor which is believed to derive from the Sertoli cells and which has the potential to exert regulatory effects on Leydig cell testosterone secretion is ‘testicular LHRH’ (see Sharpe, 1984 for review), although attempts to demonstrate its physiological importance in the normal rat testis have met with little or no success (Huhtaniemi et al., 1984a,b; Sharpe and Cooper, 1984b). More recently, we have identified the presence within the testis of a non-gonadotrophic polypeptide factor (or factors), which can exert major stimulatory effects on Leydig cell testosterone secretion (Sharpe and Cooper, 1984a). This factor is present in the testicular interstitial fluid which bathes the Leydig cells and seminiferous tubules, and its levels have been shown to change significantly in situations in which marked changes occur in testosterone levels and/or Sertoli-Leydig cell interaction (Sharpe and Cooper, 1984a; Sharpe and Bartlett, 1985). To shed more light on the possible physiological role and mechanism of action of this polypeptide factor(s), we have compared its actions and interactions with those of an LHRH agonist and with hCG using in vitro incubations of Percoll-purified rat Leydig cells. Materials and methods Animals Animals were from our own colony of Sprague-Dawley rats and were housed under conventional conditions. For preparation of Leydig cells, rats aged 80-90 days were used whilst animals aged 80-150 days were used for the bulk preparation of testicular interstitial fluid. Collection and preparation of testicular interstitial fluid (IF) Testicular IF was collected from the testes of 40-60 rats over 16-20 h at 4°C as described previously (Sharpe and Cooper, 1983, 1984a). Following centrifugation, the IF from individual testes was pooled and extracted twice with 20 mg/ml charcoal (Norit A; Sigma) for 1 h at 21°C to remove steroids; this procedure removes > 98% of

the testosterone present in IF (Sharpe and Cooper, 1984a). The extracted IF was then stored in aliquots at -40°C until used for the experiments described below. In one experiment, IF obtained from the testes of B-week bilaterally cryptorchid rats was used, after charcoal extraction as described above, and this preparation was approximately 5 times more potent than IF from control testes in stimulating Leydig cell testosterone production. Leydig cell preparation and incubation Leydig cells were isolated from the testes of groups of 10 rats and were purified on discontinuous gradients of Percoll (Pharmacia) as described previously (Sharpe and Fraser, 1983). Aliquots of 0.05 X lo6 cells, of which 80-90s were histochemically identifiable as Leydig cells (Sharpe and Fraser, 1983), were then incubated, with the additions described below, in plastic multiwell dishes (Nunc, Denmark) for 5 h at 32°C under a humidified atmosphere of 95% air:5% COZ. The medium used for incubation was medium 199 to which was added Earle’s salts, sodium bicarbonate (0.22%) L-glutamine (2 mM; Flow Laboratories), transferrin (5 pg/ml; Sigma), insulin (10 pg/ml; Sigma), ceruloplasmin (1 U/ml; Sigma), penicillin (100 W/ml; Flow Labs.), streptomycin (100 pg/ml; Flow Labs.), fungizone (2.5 pgg/ml; Flow Labs.) and BSA (0.5 mg/ml). The final incubation volume was 250 pi/well, except in time-course experiments in which the initial volume was between 300 and 500 ~1 and from which 50 ~1 aliquots were removed at intervals during incubation. Cells were incubated for periods of a-24 h, depending on the nature of the experiment. At the end of incubation, the medium was aspirated and stored at -20°C before measurement of testosterone. Two groups of experiments were performed. The first of these compared the stimulator-y effect of testicular IF on testosterone production with that of hCG and an LHRH agonist ((D-Ser-tbu6,des-Gly-NHi’)LHRH ethylamide; Hoechst), alone or in combination. The LHRH agonist was added at a concentration (4 ng/ml) in excess of that required to maximally stimulate testosterone production (Sharpe and Cooper, 1982a), whilst the IF was added at a concentration (20%) which

249

would elicit approximately the same degree of stimulation as the LHRH agonist. The ability of an excess (1 pg/ml) of an LHRH antagonist (( Nacetyl-de-Pro13’4rpFD,D-Trp3*6)LHRH; Universal Biologicals) to block the stimulatory effects of IF and the LHRH agonist on testosterone production was also assessed. These studies were performed either in the absence of any added gonadotropin or in the presence of a supra-maximally stimulating concentration (5 nM) of hCG (Chorulon, Intervet), and the cells were incubated for 5 or 20 h. In a separate experiment the effects of the same hormone additions on the rate of testosterone production were assessed at various time intervals between 2 and 20 h. For this experiment 50 ~1 of the incubation medium was removed at the desired intervals and added to 200 ~1 medium before storage at -20°C for assay. In this way the concentration of hormones and IF was kept constant in the incubation medium and both the accumulation and rate of testosterone production could be assessed. Under the conditions described, the change in the incubation volume from 500 ~1 (at time zero) to 250 ~1 (at 20 h) made no difference to the response of the cells over this time period (unpublished data). The second group of experiments characterized the ability of testicular IF to enhance hCG-stimulated testosterone secretion. Initially, the effect of incubation of Leydig cells with 20% IF for 4, 2 or 24 h on the rate of testosterone production at intervals over 2-24 h was assessed, to determine whether the IF needed to be present continuously to elicit increased testosterone production. In this study, the medium (350 ~1) from control (hCG alone) wells and those containing IF + hCG was replenished at f and 2 h, whilst sampling at 6 and 12 h was achieved by removing 50 ~1 of the incubation medium as described above. A second experiment assessed the effect of a constant concentration of testicular IF (30%) on testosterone production over 4 or 20 h by Leydig cells exposed to various concentrations of hCG. In a follow-up experiment, the effect of IF on the rate of testosterone secretion over 4 h was assessed using Leydig cells which had been preincubated for 4 h in the presence of various concentrations of hCG. Finally, it was determined whether testicular IF

had dose-dependent effects on the rate of hCGstimulated testosterone production by Leydig cells during short (O-5 h) and longer (5-20 h) term incubation. Cells were incubated in 300 ~1 for 20 h in the presence of a supra-maximally stimulating concentration (5 nM) of hCG to which were added increasing concentrations (1.2-16.7%) of charcoalstripped testicular IF collected from the testes of bilaterally cryptorchid rats, and which was approximately 5 times more potent that IF from control testes at stimulating testosterone production (data not shown, but see Sharpe and Cooper, 1984a). Testosterone production at 5 h was determined in 50 ~1 of medium removed from each well. Testosterone measurement Testosterone in unextracted media was measured by radioimmunoassay (Corker and Davidson, 1978) after appropriate dilution, as described previously (Sharpe and Cooper, 1983). Statistical analysis Results were analysed using 2-factor analysis of variance (with replication) and Student’s t-test. Results

Comparison of the sport-term (5 h) stimulator effects of testicular interstitial fluid (IF), hCG and an LHRH agonist on testosterone production Incubation of Leydig cells in the presence of 4 ng/ml LHRH agonist caused a 5-fold increase in testosterone production over basal values, an effect that was blocked completely by co-incubation with an LHRH antagonist (Fig. 1, left). Addition of 20% testicular IF to cells caused a 4-fold increase in testosterone production, but this effect was not blocked by co-~cubation with an LHRH antagonist (Fig. 1, left). Incubation of Leydig cells in the presence of both IF and the LHRH agonist resulted in significantly greater (P < 0.001) testosterone production than with either compound alone, and their combined effects appeared to be additive (Fig. 1, left). Incubation of Leydig cells for 5 h in the presence of 5 nM hCG resulted in more than a 27-fold increase in testosterone production over basal values and co-incubation with LHRH agonist caused

250 INCUBATED

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Fig. 1. Comparison of the effects of hCG (5 nM), an LHRH agouist (LHRH-A; 4 ng/ml) or testicular interstitial fluid (IF; 20%) alone or in combination, on testosterone production by isolated Leydig cells during 5 h incubation. Each value is the mean f SD of triplicate incubations, and comparable results were obtained in a repeat experiment. LHRH ANT = LHRH antagonist (1 gg/ml). It should be noted that in this and subsequent figures, the concentrations used of hCG and LHRH agonist were maximally stimulating whereas the concentration of IF used was always submaximally stimulating (see text).

a further small, but statistically insignificant (P > 0.05), increase in testosterone output; the latter effect was prevented by addition of an LHRH antagonist (Fig. 1, right). Addition of 20% IF to hCG-stimulated cells caused significant enhancement of testosterone production compared to hCG alone, and this increase was not blocked by addition of an LHRH antagonist (Fig. 1, right). Incubation of cells in the presence of hCG + IF + LHRH agonist resulted in significantly greater (P < 0.05) testosterone production than with any of the 2 compounds alone (Fig. 1). Comparable results were also obtained after 20 h of incubation, namely that any effects of LHRH agonist on testosterone production were blocked by co-in-

cubation of cells with an LHRH antagonist, whereas this compound had no effect on IFstimulated testosterone production (data not shown). However, at 20 h the relative effects of hCG, IF and LHRH agonist on testosterone production were considerably different from those seen at 5 h of incubation (data not shown), and to examine this more closely a detailed study of the temporal effects of these compounds was made. Temporal effects of testicular interstitial fluid (IF), hCG and an LHRH agonist on testosterone production Addition of LHRH agonist, IF or hCG alone to Leydig cells, caused significant (P < 0.001) stimu-

251

j-2

2-415

4-5-6

INCUBATION HOURS

OF INCUBATION

6-20 PERIOD

(h)

Fig. 2. Comparison of the temporal effects of hCG (5 nM), an LHRH agonist (LHRH-A; 4 ng/ml) or testicular interstitial fluid (IF; 20%). alone or in combination, on cumulative testosterone production by isolated Leydig cells. Each value is the mean f SD of triplicate incubations.

Fig. 3. Comparison of the effects of hCG (5 nM), an LHRH agonist (LHRH-A; 4 ng/ml) or testicular interstitial fluid (IF; 20%). alone or in combination, on the rate of testosterone production by isolated Leydig cells at different time intervals. Each value is the mean f SD of triplicate incubations, and the data is derived from the expt. illustrated in Fig. 2.

lation of testosterone production over 20 h of incubation, although the magnitude of these effects compared to basal testosterone production varied considerably between the compounds (Fig. 2). Moreover, whereas the stimulatory effect of hCG on testosterone production was clearly evident by 1 h of incubation the effects of either IF or LHRH agonist alone first became evident at 3-4: h (Fig. 2). Co-incubation of cells with hCG and LHRH agonist resulted in a small but consistent enhancement of testosterone production over 2-6 h when compared with hCG alone (P -c 0.01 in comparison of the 2-6 h period), but by 20 h the LHRH agonist had caused a small but insignificant (P > 0.05) decrease in steroid production compared to hCG alone (Fig. 2). Addition of IF to cells exposed to LHRH agonist caused consistent significant enhancement of testosterone production over 4$-20 h, when compared with LHRH agonist alone, but the most spectacular effect of IF was on hCG-stimulated testosterone production. Addition of IF significantly enhanced

hCG-stimulated testosterone production from 4; to 20 h, and the magnitude of this effect increased progressively with time (Fig. 2); further addition of LHRH agonist to such cells had no detectable effect. As the effects of the 3 compounds tested altered significantly with time, the data were also plotted as the testosterone production rate over the various time intervals (Fig. 3), and this demonstrated certain key facts. First, that either LHRH agonist or IF alone did not increase the rate of testosterone production until beyond 2 h, and whereas IF then maintained an increased rate of testosterone production up to 20 h, the stimulatory effect of the LHRH agonist disappeared beyond 6 h. Second, incubation of cells with IF + LHRH agonist had an additive stimulator-y effect on the testosterone production rate over 2-6 h, but between 6 and 20 h the rate returned to levels seen with IF alone, presumably because of the loss of the stimulatory effect of the LHRH agonist. Third, addition of hCG significantly enhanced (P -c0.001) the rate

252

of testosterone production at all time intervals, but the magnitude of this effect decreased progressively beyond 43 h. Fourth, addition of LHRH agonist to the hCG caused only a minor increase in the rate of testosterone production up to 6 h, when compared to hCG alone, but between 6 and 20 h it significantly reduced (P < 0.001) the rate of hCG-stimulated testosterone production. Finally, the addition of IF to hCG had no significant effect on the testosterone production rate up to 2 h, but thereafter caused a significant increase in the rate compared to hCG alone, and from 2 to 20 h m~ntained a high and almost constant rate of testosterone production. In other similar experiments it was confirmed that the stimulatory effect of hCG on the rate of testosterone production declined progressively with time whereas addition of IF to the hCG either attenuated or prevented this decline (data not shown, but see also Fig. 4). It should also be noted that whilst the absolute rate of testosterone production varied considerably between experiments, the magnitude and pattern of effect of IF remained relatively constant.

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Effect of interrupted exposure to testicular interstitial fluid (IF) on hCG-stimulated testosterone production Because of the relatively slow onset of the stimulatory effects of IF on the testosterone secretion rate, the duration of exposure to IF necessary to induce stimulation was assessed (Fig. 4). Exposure of Leydig cells to hCG + IF for only 15 ruin induced an increase in the rate of testosterone production over O-2 h, when compared to hCG alone, but at subsequent time points up to 24 h no difference from hCG alone was evident. Exposure to IF for 2 h resulted in significant enhancement of the rate of testosterone secretion up to 12 h, when compared to hCG alone, but this difference had disappeared at 12-24 h. Moreover, when compared with cells exposed continuously to hCG f IF, the rate of testosterone secretion by cells exposed to IF for only 2 h was massively reduced (P < 0.001) at 2-24 h (Fig. 4). These results suggest that prolonged or continuous exposure to IF is necessary to maintain greatly increased rates of testosterone secretion.

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Fig. 4. Temporal changes in the testosterone production rate by Leydig cells incubated in the presence of hCG (5 nM) alone, or hCG + testicular interstitial fluid {IF; 20%) for $, 2 or 20 h. Note that, with the exception of baseline samples, hCG was present throughout the experiment in all other incubates. Each value is the mean f SD of triplicate incubations.

The effect of testicular interstitial fluid (IF) on testosterone secretion induced by different doses of hCG When a constant amount of testicular IF was added to cells stimulated with a wide range of hCG doses during 4 or 20 h incubation, the resultant enhancement of testosterone production by the IF was not related strictly to the dose of hCG but to the magnitude of hCG-stimulated testosterone production (Table 1). Thus, doses of hCG ranging from 5 to 50000 pM all caused maximal stimulation of testosterone production, and addition of IF caused comparable enhancement of steroid output for all of these doses. In contrast, although addition of IF to doses of hCG (0.05 and

253

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disparity was evident at both 4 and 20 h of incubation, it was more exaggerated at the later time (Table 1). Comparable effects were also noted in experiments in which cells were preincubated for 2 or 4 h in the presence of different hCG doses and then stimulated for a further 4 h with hCG + IF (data not shown). Effect of different doses of testicular interstitial fluid (IF) on the testosterone production rate When increasing doses of testicular IF were added to cells exposed to a supra-maximally stimulating concentration (5 nM) of hCG, the IF caused dose-dependent stimulation of the rate of testosterone production over both O-5 and 5-20 h, with rates over the latter time period always less than those over O-5 h (Fig. 5).

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Fig. 5. Dose dependency of the stimulatory effects of testicular interstitial fluid on the rate of testosterone production by isolated Leydig cells stimulated maximally with 5 nM hCG. Note that the interstitial fluid used in this experiment was obtained from cryptorchid rat testes (see text) and was approximately 5 times more potent than the preparations from normal testes used in Figs. 1-4. Each value is the mean +SD of triplicate incubations.

0.5 PM) which caused submaximal testosterone production still enhanced steroid secretion, the effects were proportionately much smaller than those seen at higher hCG doses. Although this TABLE

The present findings confirm and extend our previous observations (Sharpe and Cooper, 1984a) in showing that interstitial fluid from the normal adult rat testis can exert major stimulatory effects on Leydig cell testosterone secretion. In its mode of action, IF appears to be different from both hCG and LHRH agonist and has the ability to interact with either of these hormones. These facts coupled with the magnitude of the stimulatory effects of IF on testosterone secretion all argue that the causative factor(s) in testicular IF may play an important physiological role, and may also

1

COMPARISON TESTOSTERONE hCG Concentration of hCG (PM)

OF THE MAGNITUDE OF THE EFFECT OF TESTICULAR INTERSTITIAL FLUID PRODUCTION OVER 4 AND 20 h BY LEYDIG CELLS STIMULATED WITH A RANGE

Testosterone

production

(ng/106

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Discussion

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***

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incubation

in the absence

of IF.

IF-induced increment ** *** *** ***

311 4562 3682 4 849 614 280

254

provide new insight into the mechanisms involved in controlling testosterone biosynthesis. In certain respects the actions of testicular IF and an LHRH agonist on testosterone production were similar whilst in others they differed greatly. In general, both IF and LHRH agonist took much longer (l-3 h) to initiate an increase in testosterone secretion than did hCG ( < 1 h), and whilst this may be partly a consequence of the lower levels of stimulation achieved by these compounds on their own, when compared with hCG, a definite lag phase in the action of IF was also usually evident in the presence of hCG (e.g. Fig. 3). A further point of similarity was that both IF and the LHRH agonist increased testosterone production in the presence of ma~m~ly stimulating concentrations of hCG, although in the present experiments this effect of LHRH agonist was poor in relation to previous results (Hunter et al., 1982; Sharpe and Cooper, 1982a). Lastly, to maintain its stimulatory effect on testosterone production, IF had to be present continuously or for prolonged periods, and this is very similar to the mode of action of LHRH and its agonists (Sharpe and Cooper, 1982b). In all other respects the effects of IF and LHRH agonist on testosterone production were quite different. In particular, the degree of stimulation of testosterone production elicited by IF was considerably greater than could be achieved with a maximally stimulating concentration of LHRH agonist, and this difference was particularly marked in the presence of hCG. Again, whilst the stimulatory effects of IF appeared to increase (in the presence of hCG) or plateau (in the absence of hCG) with increase in the time of incubation, the stimulator effects of LHRH agonist did not persist beyond 6 h, with evidence of inhibition appearing by 20 h (Fig. 3), as has been reported previously by other authors (Hunter et al., 1982; Browning et al., 1983). These differences suggest that the active peptide in IF is not a macromolecular LHRHlike peptide, and conclusive proof of this was provided by the inability of an LHRH antagonist to block the stimulatory effects of IF. The observation that IF could enhance the testosterone response to LHRH agonist, to hCG and also to hCG + LHRH agonist, argues that the IF exerts its stimulatory effect on testosterone secretion via mechanisms separate from those of

LHRH agonist and hCG. As the former appears to exert its effects via cyclic AMP-independent pathways (e.g. Sullivan and Cooke, 1983, 1984), it implies that this messenger is not involved in the actions of IF. Additional, if indirect, support for this conclusion is provided by the observation that IF enhanced testosterone production equally in the presence of a wide range of ma~mally stimulating concentrations of hCG (Table l), which might be expected to produce stepwise increases in cyclic AMP output (Dix and Cooke, 1981). Other evidence to this effect is that even when Leydig cell testosterone secretion is stimulated by mechanisms which bypass cyclic AMP (e.g. addition of 1 PM hydroxycholesterol), then addition of IF can still enhance testosterone output (I. Cooper and R.M. Sharpe, unpublished data). An important difference between the effects of IF on LHRH agonist-stimulated, as opposed to hCG-stimulated, testosterone production was that in the former situation the effect of IF was purely additive, whereas in the presence of maximally stimulating concentrations of hCG the effect of IF was clearly synergistic. However, in the presence of submaximally stimulating concentrations of hCG the effect of IF was proportionally much smaller than was its effect in the presence of maximally stimulating doses of HCG (Table 1). This suggests that perhaps the most important factor determining the magnitude of the stimulatory effects of IF is the degree of stimulation of testosterone production by other hormones. Whilst the present data on IF effects add weight to the belief that important intratesticular mechanisms exist to enable local control of testosterone secretion, it should not be forgotten that Leydig cells are exposed continuously to IF in vivo. It could therefore be argued that the stimulatory effects of IF observed in vitro are just a consequence of restoring the normal ‘stimulatory’ environment. Indeed, the present finding that addition of IF to Leydig cells for 2 h followed by its removal resulted in near cessation of its stimulatory effect, is consistent with the view that the process of isolating the cells results in detrimental changes in steroidogenic responsiveness due to removal of one or more stimulatory factors present in the IF. This interpretation in no way devalues the potential regulatory importance of the IF fac-

255

tar(s) in question, particularly as their level in IF has been shown to change significantly and meaningfully in certain experimental situations (Sharpe and Cooper, 1984a; Sharpe and Bartlett, 1985). However, if this interpretation is correct, then it has major implications for all investigators who routinely isolate rat Leydig cells and study their ‘normal’ function in vitro. Acknowledgements

I am grateful to Irene Cooper for her skilled help and to Dr. Jurgen Sandow and Hoechst for the supply of LHRH agonist. References Bergh, A. (1983) Int. J. Andrology 6, 57-65. Browning, J.Y., D’Agata, R., Steinberger, A., Grotjan Jr., H.E. and Steinberger, E. (1983) Endocrinology, 113, 985-991. Corker, C.S. and Davidson, D.W. (1978) J. Steroid Biochem. 9, 373-374. de Kretser, D.M. (1982) Int. J. Andrology Suppl. 5, 11-17. Dix, C.J. and Cooke, B.A. (1981) B&hem. J. 196, 713-719. Huhtaniemi, LT., Stewart, J.M., Channabasavaiah, K., Fraser, H.M. and Clayton, R.N. (1984a) Mol. Cell. Endocrinol. 34, 127-135. Huhtaniemi, LT., Stewart, J.M., Channabasavaiah, K., Fraser, H.M. and Clayton, R.N. (1984b) Mol. Cell. Endocrinol. 34, 137-143.

Hunter, M.G., Sullivan, M.H.F., Dix, C.J., AIdred, L.F. and Cooke, B.A. (1982) Mol. Cell. Endocrinol. 27, 31-44. Kerr, J.B., Rich, K.A. and de Kretser, D.M. (1979) Biol. Reprod. 20, 409-422. Risbridger, G.P., Kerr, J.B. and de Kretser, D.M. (1981) Biol. Reprod. 24, 534-540. Sharpe, R.M. (1983) Q.J. Exp. Physiol. 68, 265-287. Sharpe, R.M. (1984) Biol. Reprod. 30, 29-49. Sharpe, R.M. and Bartlett, J.M.S. (1985) J. Reprod. Fertil. (in press). Sharpe, R.M. and Cooper, I. (1982a) Mol. Cell. Endocrinol. 26, 141-150. Sharpe, R.M. and Cooper, I. (1982b) Mol. Cell. Endocrinol. 27, 199-211. Sharpe, R.M. and Cooper, I. (1983) J. Reprod. Fertil. 69, 125-135. Sharpe, R.M. and Cooper, I. (1984a) Mol. Cell. Endocrinol. 37, 159-168. Sharpe, R.M. and Cooper, I. (1984b) In: Hormonal control of the hypothalamo-pituitary-gonadal axis, Ed.: K.W. McKerns (Plenum Press, New York) pp. 455-466. Sharpe, R.M. and Fraser, H.M. (1983) Mol. Cell. Endocrinol. 33,131-146. Sharpe, R.M., Cooper, I. and Doogan, D.G. (1984) J. Endocrinol. 102, 319-327. Sullivan, M.H.F. and Cooke, B.A. (1983) B&hem. J. 216, 747-752. Sullivan, M.H.F. and Cooke, B.A. (1984) Mol. Cell. Endocrinol. 36, 115-122.