hCG by cryptorchid testes was reduced at all times after injection by up to 70%. The ... such as cryptorchidism, X-irradiation, hydroxyurea treatment or vitamin A ...
Impaired gonadotrophin uptake in cryptorchid rat testis R. M. M.R.C.
vivo
by
the
Sharpe
Reproductive Biology Unit, Centre For Reproductive Biology, Edinburgh EH3 9EW, U.K.
37 Chalmers Street,
Summary. The specific testicular uptake in vivo of 125I-labelled hCG was compared in control adult rats and adult rats made bilaterally cryptorchid 5 weeks previously. Although a similar temporal pattern of uptake was observed in both groups, uptake of hCG by cryptorchid testes was reduced at all times after injection by up to 70%. The possible causes of this impairment were investigated. It could not be accounted for by differences in the rate of absorption or clearance of 125I-labelled hCG in the two groups. Therefore, because hCG-induced increase in the permeability of testicular capillaries is a crucial factor in determining hCG uptake by the testis, this change was compared in control and cryptorchid testes. Although hCG induced a characteristic increase in testicular capillary wall permeability in both groups, this change was temporally delayed in cryptorchid testes, and occurred after hCG values in the blood had fallen. Even when hCG had crossed the capillary wall into testicular interstitial fluid, its uptake into the testicular tissue was significantly lower in cryptorchid than in control testes. These changes probably account for the impairment of gonadotrophin uptake by the cryptorchid testis and have important implications with respect to the aetiology of Leydig cell changes in cryptorchidism. Introduction The induction of seminiferous tubule damage in adult rats by a variety of experimental techniques such as cryptorchidism, X-irradiation, hydroxyurea treatment or vitamin A deficiency, results in marked changes in Leydig cell morphology and function (Kerr, Rich & de Kretser, 1979 ; Rich & de Kretser, 1979; de Kretser, Sharpe & Swanston, 1979). Typically, there is an increase in Leydig cell volume and hypertrophy of the cellular organdíes involved in steroidogenesis (Kerr et al, 1979; Rich, Kerr & de Kretser, 1979). Surprisingly, despite these changes and the raised circulating levels of luteinizing hormones (LH) characteristic of such animals, the serum levels of testosterone are generally low to normal, and the testosterone response to injected LH or human chorionic gonado¬ trophin (hCG) is generally subnormal (Kerr et al, 1979 ; Rich & de Kretser, 1979 ; Rich et al., 1979 ; Damber, Bergh & Janson, 1980; Schanbacher, 1980b) although it is increased if enormous doses of hCG are injected (Risbridger, Kerr, Peake, Rich & de Kretser, 1981b). Paradoxically, when the maximal steroidogenic response of testicular tissue from rats with damaged seminiferous tubules is assessed in vitro (by incubation with hCG), the amounts of testosterone secreted are always 2-4 times greater than in controls (de Kretser et al., 1979; Rich & de Kretser, 1979; Schanbacher, 1980a). This difference between in-vivo and in-vitro findings is unexplained. It has been shown that hCG (LH) regulates the permeability of testicular capillaries (Setchell & Sharpe, 1981), and this is an important factor in determining how much gonadotrophin reaches the Leydig cells to be taken up onto LH (hCG) receptors (Sharpe, 1981). Failure of this mechanism 0022-4251 /83/020379-09S02 00/0 © 1983 Journals of Reproduction & Fertility Ltd
would result in decreased transport of gonadotrophin to the Leydig cells and hence 'apparent' impairment of testosterone responsiveness to gonadotrophin in vivo, but not in vitro because the mechanism would be irrelevant under these conditions. Impairment of capillary wall permeability in the cryptorchid testis would thus neatly account for the opposition of in-vivo and in-vitro findings detailed above. This paper explores this possibility.
Materials and Methods Animals and treatments. Inbred PVG male rats aged 80 days were obtained from Bantin and Kingman Ltd, Hull, and were maintained under standard conditions. Bilateral cryptorchidism was induced in 36 rats under ether anaesthesia by translocating the testes to the abdominal cavity and suturing the inguinal canal to prevent their redescent into the scrotum. A group of 36 shamoperated rats served as controls. At 5 weeks after operation, rats were injected with a total of 45-5 i.u. hCG (Pregnyl), which included either 125I-labelled bovine serum albumin (BSA; 3-70 IO6 c.p.m.) or 0-5 i.u. 125IIO6 c.p.m.); injections were given subcutaneously in 0-2 ml 0-9% labelled hCG (CR 115; 2-85 (w/v) NaCl containing 0-2% (unlabelled) BSA. Groups of 3 rats in each treatment group were killed with C02 at 2,4,8,16,24 and 40 h after injection and subjected to the procedures described below. Collection of samples and measurement of radioactivity. The methods used were essentially the same as those described previously (Sharpe, 1980, 1981). When animals were killed, blood was collected after decapitation, the serum separated by centrifugation and the ' 25I-activity in a 0-5 ml sample was determined by counting for 1 min in a gamma counter. The testes were dissected out and individually placed into preweighed 83 13 mm tubes and weighed. The caudal end of the testicular capsule was then carefully incised and the interstitial fluid allowed to drain from the testis into the tube bottom over the next 16 h at 4°C. The testes were then removed and the fluid remaining in the tubes was weighed and its 125I content measured. The 125I activity remaining in the testis (i.e. associated with 'testis tissue') was determined by placing each testis into an 83x13 mm tube and counting in a gamma counter. 'Testis tissue' weight was calculated by subtracting the weight of interstitial fluid from the originally recorded testis weight. Protein-associated 125I-activity in serum and interstitial fluid from rats injected with hCG + 125I-labelled hCG was determined by trichloroacetic acid (TCA) precipitation of pooled material from the animals at each time point. An equal volume of 10% TCA was added to duplicate tubes containing 0-5 ml serum or 0-2 ml interstitial fluid. The tubes were vortexed vigorously then centrifuged for 15 min at 1500 g and the 125I activity in the supernatant and precipitate was determined. Uninjected 125I-labelled hCG added to 0-5 ml rat serum was subjected to the same process for comparison. Radioiodination of hCG and BSA. The hCG (CR 115; 13 200 i.u./mg) and BSA (fraction V, Sigma) were labelled with 125I using lactoperoxidase (Miyachi, Vaitukaitis, Nieschlag & Lipsett, 1972) to specific activities of 56 and 53pCi/pg, respectively. The 125I-labelled hCG showed in excess of 46% specific binding to isolated Leydig cells in vitro, and, although this particular preparation was not tested, 125I-labelled hCG prepared in this manner consistently retains full biological activity in vitro (Sharpe, Fraser & Sandow, 1979). Assessment of the specific testicular uptake of12SI-labelled hCG in vivo. In the present experiment the level of 125I-labelled hCG in testicular tissue (i.e. after removal of the interstitial fluid) in rats injected with hCG + 125I-labelled hCG is compounded by uptake onto hCG receptors and non¬ specific 'uptake'; the latter includes ' 25I-labelled hCG in any residual interstitial fluid and in blood within the testis as well as non-receptor-bound but tissue-associated 125I-labelled hCG and/or degradation products. Ideally, non-specific binding should be determined by administering a large excess of unlabelled hCG. However, hCG has dose-related effects on capillary wall permeability and testicular lymph flow (Setchell & Sharpe, 1981) and these factors strongly influence hCG
'uptake' (Sharpe, 1981), so that a valid measure of non-specific binding is impossible to obtain. Therefore, a different approach has been adopted based on the relative testicular levels of 125Ilabelled BSA in comparable rats injected with the same total dose of hCG but including 125Ilabelled BSA. These values have been used to correct for non-specific uptake in rats injected with
hCG + 125I-labelledhCG. Thus, in a rat injected with hCG + 12SI-labelledhCG and killed at time T, and in which the serum ' 25I-labelled hCG level in c.p.m./mg S, the testicular tissue weight in Wand the testicular tissue content of 125I-labelled hCG in c.p.m./testis H, the specific mg =
=
=
binding (in c.p.m./testis)
was
computed
- \WxSx
as:
125I-labelled BS A/mg testis tissue at time c.p.m. 125I-labelled BSA/mg serum at time
c.p.m.
~\
J
The correction factor (i.e. the testis tissue/serum 125I-labelled BSA ratio) ranged between 0-08 and 010 for control rats and between 0-21 and 0-29 for cryptorchid rats. These values remained virtually constant with time and at 16 h, when testicular 125I-labelled hCG and 125I-labelled BSA levels were at their highest, represented 14 and 37% respectively of the comparable uncorrected values for 12SI-labelled hCG in control and cryptorchid rats. Estimation of the proportional uptake of12SI-labelled hCGfrom testicular interstitialfluid. This was assessed as described previously (Sharpe, 1981) and is based on the principle that as BSA is of a molecular size similar to hCG, it will pass from the bloodstream into testicular interstitial fluid at a rate similar to that of hCG, but it is not taken up by receptors in the testis. Therefore, the interstitial fluid/serum ratio for BSA gives a measure of how much hCG would be in the interstitial fluid if there was no uptake of this hormone onto LH (hCG) receptors. The difference between the interstitial fluid/serum ratio for 125I-labelled BSA in rats injected with hCG + 125I-labelled BSA and that for 125I-labelled hCG in rats injected with hCG + 125I-labelled hCG thus provides a measure of what proportion of the hCG in interstitial fluid has been taken up onto hCG receptors. The statistical significance of results was assessed using 2-factor analysis of variance (with replication) and Student's t test. Results Testicular tissue
weight and interstitial fluid volume
In neither of the treatment groups did testicular tissue weight vary significantly with time, but in cryptorchid rats the weight of the testes was on average only 36% (P < 0-001) ofthat in controls
(Table 1).
Table 1.
Temporal changes in interstitial fluid levels and testicular tissue weight after injection of control and bilaterally cryptorchid rats with hCG Control rats
Time after hCG (h) 2 4 8 16 24 40
Wt of paired testes
(mg)t
2770 2700 2619 2810 2834 2838
± ± ± ± ± ±
Interstitial fluid
Wt of
(mg/paired testes)
testes
177 240 288 219 207 143
68 101 190 136 246 142
Cryptorchid rats
± 33 ± 50* ± 34*** ± 19* ± 36 ± 17
906 996 1082 1136 1087 1055
paired (mg)f ± 170 ± 140 ± 124
± 212 ± 118 ± 177
Interstitial fluid
(mg/paired testes) 310 339 406 555 516 305
± 83
± 124 ± 91 ± 74**
± 66** ± 71
Values are mean ± s.d. for 6 rats. *P < 005, **P < 001, ***P < 0001 in comparison with respective 2-h values (Student's t
test). t Weight of paired
weight
testes —
of interstitial fluid.
In control rats injected with hCG (and 125I-labelled BSA or 125I-labelled hCG), the volume of testicular interstitial fluid increased significantly (P < 0-05) between 2 and 4 h, reached a peak at 8 h ( < 0-001) and had returned to the starting level by 40 h (Table 1). In cryptorchid rats, the interstitial fluid volume was always considerably greater (P < 0-001) than in controls and hCG injection led to a significant increase (P < 0-001) in interstitial fluid levels at 16 and 24 h with some evidence of an earlier increase at 8 h; as in controls, the fluid volume had returned to starting levels by 40 h. As changes in the volume of interstitial fluid are a reliable guide to changes in capillary wall permeability (Setchell & Sharpe, 1981), these results suggest that hCG is capable of increasing capillary wall permeability in testes from control and bilaterally cryptorchid rats, but that in the latter this increase is considerably delayed compared with controls.
Specific uptake ofl2SI-labelled hCG by the testis The pattern of specific uptake of 125I-labelled hCG by testicular tissue was similar in both groups, binding increasing up to 16 h and then declining by 40 h (Text-fig. 1). However, at each of the times studied, binding to control rat testes was always higher ( < 0001) than to testes from cryptorchid rats and this difference was particularly marked at 16-40 h when more than a 3-fold difference was evident. A similar difference (P < 0-001) was observed when comparing values for the binding of 125I-labelled hCG which were uncorrected for non-specific binding, although the magnitude of difference between uptake by control and cryptorchid rat testes was less marked because of the higher 'non-specific uptake' (i.e. testicular tissue levels of 125I-labelled BSA) in cryptorchid rat testes.
Control
151
/ï\ \
/
\
'/ /y 2 4
8
Bilaterally cryptorchid
--_
-Í 16
24
40
Hours after injection
Text-fig. 1. Temporal changes in the specific testicular uptake of 125I-labelled hCG in control and bilaterally cryptorchid rats after a single subcutaneous injection of 45 i.u. unlabelled hCG + 0-5 i.u. 125I-labelled hCG. Each point is the mean ± s.d. for 3 animals. Serum levels and metabolism
ofX2iI-labelled hCG
None of the preceding differences in the testicular uptake of 125I-labelled hCG could be explained by alteration of the rate of absorption or clearance of the labelled hormone (Text-fig. 2). In control and cryptorchid rats the serum levels of 125I-labelled hCG increased between 2 and 8 h and then declined slowly but progressively up to 40 h. Similarly, there was no difference between the two groups in the absorption and clearance of 125I-labelled BSA following injection of un¬ labelled hCG and 125I-labelled BSA (Text-fig. 2).
Cryptorchid
Control 60
50
40< (
30-
20-
10-
Serum Interstitial fluid
Hours after
injection
Text-fig. 2. Temporal changes in the concentration of ' 25I-labelled BSA and ' 25I-labelled hCG in serum and testicular interstitial fluid after injection of control and bilaterally cryptorchid rats with a total of 45-5 i.u. hCG and which included 125I-labelled BSA (top) or 125I-labelled hCG (bottom). Each point is the mean + s.d. for 3 animals.
injected with hCG + 125I-labelled hCG the serum 125I levels reflected predominantly protein-associated 125I as judged from TCA precipitation (Text-fig. 3), although there was a clear increase with time in the proportion of TCA-soluble 125I activity. However, this change was equally evident in control and cryptorchid rats. In contrast, the TCA-precipitable 125I activity in interstitial fluid was always lower than that in the corresponding serum sample, a difference that was particularly marked in the first 8 h (Text-fig. 3). This coincided with the period when the proportionate uptake of ' 25I-labelled hCG from interstitial fluid was at its peak (see below) and it is this selective removal of intact (i.e. protein-associated) 125I-labelled hCG onto receptors that is primarily responsible for increasing the relative proportion of TCA-soluble 125I activity (see Sharpe, 1980,1981). In keeping with this view, at 2-8 h interstitial fluid levels of 125I-labelled hCG in both groups were always much lower than the corresponding serum values, a difference not found to the same extent in animals injected with 125I-labelled BSA (Text-fig. 2, but details given below). In rats
of the proportionate uptake ofl2SI-labelled hCG from interstitial fluid For control and cryptorchid rats, the ratio of the concentration of 125I activity in interstitial fluid to that in serum in rats injected with 125I-labelled BSA or 125I-labelled hCG was calculated (Text-fig. 4). As these two labels were injected with the same total dose of hCG, they are exactly comparable in terms of any changes, e.g. of capillary permeability. Therefore, the interstitial fluid : serum ratio of 125I-labelled BSA (an inert protein) can be used to calculate what concentraEstimation
Uninjected 125l-labelled hCG
2
4
8
16 24 40
2
4
8
16 24 40
Controls
Bilaterally cryptorchid Hours after injection Text-fig. 3. Temporal changes in TCA-precipitable radioactivity in serum (open bars) and testicular interstitial fluid (hatched bars) from control and bilaterally cryptorchid rats after a single injection of 45 i.u. unlabelled hCG + 0-5 i.u. 125I-labelled hCG. Results are shown as the mean of duplicate determinations for pooled material from 3 rats at each time, and duplicates agreed within 7%.
tion of hCG should be present in interstitial fluid at a given time. The difference between this theoretical value and the observed interstitial fluid : serum ratio of 125I-labelled hCG thus gives an estimation of what proportion of the 125I-labelled hCG has been taken up onto receptors. Using these values the proportionate uptake of hCG at different times after injection was calculated (Table 2) ; these values only indicate what percentage of hCG in interstitial fluid has been removed at any one time, they do not provide information on the total amount of hCG taken up over a period of time (see Sharpe, 1981). Table 2. Estimation of the proportional tes¬ ticular uptake of hCG from interstitial fluid at different times after injection of the hormone
Time after
injection (h) 2 4 8 16 24 40
% of hCG content removed by testicular uptake Control 31-4 59-9 61-7 37-4 14-4 2-6
± ± ± ± ± +
5-8 21 1-6 5-2 40 40
Cryptorchid 58-2 40-2 500 16-5 14-9 13-8
± 4-7** ± 6-5** ± 2-9** ± 1-0** ± 3-0 ± 3-4*
Values are mean ± s.d. for 3 rats at each time were derived from the ratios shown in Textfig. 4 (see text for details). *P < 005, **P < 001, in comparison with values for respective control group (Student's t
and
test).
Text-fig. 4. Temporal changes in the interstitial fluid to serum ratio of 125I activity after injection of control and cryptorchid rats with a total of 45-5 i.u. hCG which included 125Ilabelled BSA or 125I-labelled hCG. Each point is the mean ± s.d. for 3 rats and values were derived from the data illustrated in Text-fig. 2. The shaded area indicates the difference between the ratios for 125I-labelled BSA and ' 25I-labelled hCG and provides a measure of the proportionate uptake of the latter from interstitial fluid onto testicular LH (hCG) receptors (see Table 2 and text). The pattern of hCG uptake from interstitial fluid was significantly different in the two groups (Table 2). Most importantly the proportionate uptake of hCG in control rats was highest at 4-16 h which coincided (1) with the highest blood (and therefore interstitial fluid) levels of hCG (Text-fig. 2) and (2) with the maximum increase in interstitial fluid volume as a result of increased capillary wall permeability (Table 1). In contrast, hCG uptake in cryptorchid rats was significantly lower than in controls at these times, and the maximum proportionate uptake (2-8 h) preceded any major increase in capillary wall permeability. The rate at which the interstitial fluid:serum ratio for 125I-labelled BSA approaches equilib¬ rium also provides an indication of capillary wall permeability (Setchell & Sharpe, 1981), and although there was no major difference in this value between control and cryptorchid rats, values in the latter were slightly but consistently lower (P < 0-001) than in controls (Text-fig. 4). Discussion
This paper demonstrates gross impairment of gonadotrophin uptake in vivo by the cryptorchid rat testis, and this occurs despite the increased number and size of the Leydig cells (Kerr et al, 1979) and the much larger relative volume of interstitial tissue in such testes. This impairment may explain the discrepancy between previous reports which demonstrated reduced steroidogenic responsiveness of the cryptorchid rat testis in vivo but enhanced responsiveness in vitro (see 'Intro¬ duction'). It may also explain why serum levels of testosterone in the bilaterally cryptorchid rat are low to normal in the face of raised circulating levels of LH (see de Kretser et al, 1979), and it has considerable implications with respect to the aetiology of Leydig cell changes resulting from induction of cryptorchidism.
primary aim of the present investigation was to test the possibility that there was impair¬ in the cryptorchid rat testis. One manifestation of such a change would be reduced gonadotrophin uptake by the Leydig cells as a result of decreased gonadotrophin transport to the Leydig cells (Sharpe, 1981). However, no gross difference in capillary wall perme¬ ability was observed between the cryptorchid and normal testis as judged by the rate at which the interstitial fluid : serum ratio of 125I-labelled BSA reached equilibrium (Text-fig. 4), although at all time points studied the actual values for this ratio were slightly but significantly lower for cryptor¬ chid than for normal testes; this might suggest that a subtle decrease in permeability had occurred. In keeping with this observation there was a distinct difference between control and cryptorchid rats in the capillary response to hCG. Thus the hCG-induced accumulation of interstitial fluid, which reflects an increase in capillary wall permeability (Setchell & Sharpe, 1981), occurred 4-16 h after injection in control rats with a peak response at 8 h (Table 1). In contrast, this change was delayed in cryptorchid rats such that an increase in interstitial fluid volume was only just evident at 8 h with the peak response occurring at 16-24 h ; this delay may itself have resulted from the reduced uptake of hCG. Because this hCG-induced change in capillary wall permeability is of enormous importance in increasing transport of injected hCG to the Leydig cells (Sharpe, 1981), the difference in the timing of this response in control and cryptorchid rats could be a major factor in accounting for the difference in hCG uptake in these two situations. It is particularly relevant that the peak increase in capillary wall permeability in control rats coincides with the period when blood (and interstitial fluid) levels of hCG are at their highest (4-8 h), whereas the corresponding peak in capillary permeability in cryptorchid rats (16-24 h) occurs at a time when blood levels of The
ment of capillary wall permeability
hCG are declining. This delayed increase in capillary wall permeability is, however, only one factor contributing to a reduction in hCG uptake by the cryptorchid testis. Even when hCG has crossed the capillary wall into testicular interstitial fluid, its uptake from this medium is significantly reduced compared with that in controls over the period 4-16 h after injection (Table 2). Once again this deficit occurred at the crucially important time when circulating levels of hCG were highest and when the changes in capillary wall permeability were greatest. This decrease in hCG uptake from interstitial fluid may simply be a consequence of the much larger volume of interstitial fluid in cryptorchid compared with control testes, i.e. total uptake of hCG is unaffected but uptake per unit volume of interstitial fluid is reduced because of the increased fluid volume. Alternatively, the reduced uptake of hCG from interstitial fluid may be a consequence of the reported 60-70% decrease in the number of LH receptors per Leydig cell in the cryptorchid rat testis (de Kretser et al, 1979; Risbridger et al,
1981b).
The present results do not clearly resolve the aetiology of reduced gonadotrophin uptake by the cryptorchid testis, although they suggest that the cause is probably multi-factorial. All of the factors measured in this study which would influence hCG uptake were impaired in cryptorchid rats to some extent and these changes and the reported reduction in LH-receptor numbers could easily account for the decrease in hCG uptake. Although hCG has different receptor-binding characteristics and a much longer half-life than rat LH, it is not unreasonable to assume from the present findings that there is impairment of testicular uptake of endogenous LH by the cryptorchid rat testis. Indeed, the fact that serum levels of testosterone in the cryptorchid rat are low to normal in the face of chronically raised blood levels of LH (see de Kretser et al, 1979) suggests that the testes are not 'seeing' all of this LH, because such testes are capable of responding in vitro with supranormal testosterone secretion (Risbridger et al, 1981b). On the other hand, Leydig cells from cryptorchid rats show many features consistent with increased exposure to LH, e.g. hypertrophy of cellular organdíes, an increase in cell size and a loss of LH receptors (references in 'Introduction'), but these changes may not be the result of increased stimulation by LH because (1) all of the described changes occur before any increase in blood levels of LH (Risbridger et al, 1981b), and (2) exactly comparable changes occur in the abdominal, but not in the scrotal, testis of adult rats made unilaterally cryptorchid, and are not associated with any
major increase in serum LH levels (Risbridger, Kerr & de Kretser, 1981a). Instead, these Leydig cell changes are considered to be locally induced, probably involving changes in the secretion of factors from the Sertoli cells which have stimulatory or inhibitory effects on the Leydig cells (reviewed by de Kretser, 1982; Sharpe, 1982). The present findings add weight to this interpreta¬ tion as they suggest that the cryptorchid rat testis has a reduced ability to detect, and thus to respond to, the raised blood levels of LH. I
am
grateful
to
the NIAMDD,
U.S.A., for purified hCG for radioiodination. References
Damber, J.-E., Bergh,
. & Janson, P.O. (1980) Leydig cell function and morphology in the rat testis after exposure to heat. Andrologia 12, 12-19. de Kretser, D.M. (1982) Sertoli cell-Leydig cell interac¬ tion in the regulation of testicular function. Int. J. Andrei., Suppl. 5, 11-17. de Kretser, D.M., Sharpe, R.M. & Swanston, I.A. (1979) Alteration in steroidogenesis and hCG binding in the cryptorchid rat testis. Endocrinology 105, 135-139. Kerr, J.B., Rich, K.A. & de Kretser, D.M. (1979) Alter¬ ations of the fine structure and androgen secretion of the interstitial cells in the experimentally cryptorchid rat testis. Biol. Reprod. 20, 409^122. Miyachi, Y., Vaitukaitis, J.L., Nieschlag, E. & Lipsett, M.B. (1972) Enzymatic radioiodination of gonadotropins. J. clin. Endocr. Metab. 34, 23-28. Rich, K.A. & de Kretser, D.M. (1979) Effect of fetal irra¬ diation on testicular receptors and testosterone re¬ sponse to gonadotrophin stimulation in adult rats. Int. J. Androl. 1, 343-352. Rich, K.A., Kerr, J.B. & de Kretser, D.M. (1979) Evi¬ dence for Leydig cell dysfunction in rats with semini¬ ferous tubule damage. Molec. cell. Endocr. 13, 123— 135. Risbridger, G.P., Kerr, J.B. & de Kretser, D.M. (1981a) Evaluation of Leydig cell function and gonadotropin binding in unilateral and bilateral cryptorchidism: evidence for local control of Leydig cell function by the seminiferous tubule. Biol. Reprod. 24, 534-540. Risbridger, G.P., Kerr, J.B., Peake, R., Rich, K.A. & de
Kretser, D.M. (1981b) Temporal changes in Leydig cell function after the induction of bilateral cryptor¬ chidism. J. Reprod. Fert. 63, 415-423. Schanbacher, B.D. (1980a) Androgen secretion and char¬ acteristics of testicular hCG binding in cryptorchid rats. J. Reprod. Fert. 59, 145-150. Schanbacher, B.D. (1980b) Androgen response of crypt¬
orchid and intact rams to ovine LH. J. Reprod. Fert. 59, 151-154. Setchell, B.P. & Sharpe, R.M. (1981) Effect of injected human chorionic gonadotrophin on capillary perme¬ ability, extracellular fluid volume and the flow of lymph and blood in the testes of rats. J. Endocr. 91, 245-254. Sharpe, R.M. (1980) Temporal relationship between in¬ terstitial fluid accumulation and changes in gonado¬ tropin receptor numbers and steroidogenesis in the rat testis. Biol. Reprod. 11, 851-857. Sharpe, R.M. (1981) The importance of testicular inter¬ stitial fluid in the transport of injected hCG to the Leydig cells. Int. J. Andro!. 4, 64-74. Sharpe, R.M. ( 1982) The hormonal regulation of the Leydig cell. In Oxford Reviews in Reproductive Biology, Ed. C.A. Finn. Oxford University Press, Oxford (in
press). Sharpe, R.M., Fraser, H.M. & Sandow, J. (1979) Effect of treatment with an agonist of luteinizing hormonereleasing hormone on early maturational changes in pituitary and testicular function in the rat. J. Endocr. 80, 249-257.
Received 30 July 1982
