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Immuno- and bioactive inhibin and inhibin α-subunit expression in rat Leydig cell cultures
Molecular and Cellular Endocrinology, 66 (1989) 119-122 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
119
02153
Rapid Paper
Immuno- and bioactive inhibin and inhibin a-subunit Leydig cell cultures G.P. Risbridger
expression in rat
‘, J. Clements 2, D.M. Robertson I, A.E. Drummond H.G. Burger * and D.M. de Kretser ’
I, J. Muir I,
’ Department of Anatomy,Monash University, Melbourne, Victoria, Australia, and ’ Medical Research Centre, Prince Henry’s Hospital, Melbourne, Victoria, Australia (Accepted
Key words: Leydig
cell; Inhibin;
10 July 1989)
(Rat)
Summary The rise in serum immunoactive inhibin levels in male rats following hCG stimulation raised the possibility that Leydig cells may produce inhibin. This study therefore evaluated whether Percoll-purified Leydig cells from adult male rats synthesized and secreted inhibin in vitro as measured by Northern blot analysis, radioimmunoassay and in vitro bioassay. Northern blot analysis demonstrated the presence of cx-inhibin subunit mRNA in the Leydig cell and inhibin bioactivity was detected in Leydig cell culture media. Levels of immunoactive inhibin increased in culture over 20 h and were directly dependent on the number of Leydig cells in culture. rLH (NIADDK-rLH-I-6) and not rFSH (NIADDK-rFSH-I-6) stimulated immunoactive inhibin levels in a dose-depedent manner. This study demonstrates that Leydig cells express mRNA for the a-subunit of inhibin and produce inhibin which is biologically and immunologically active prompting a re-evaluation of our concepts of testicular inhibin production.
Introduction Inhibin is a glycoprotein hormone that is produced by the Sertoli cells and regulated by FSH (Steinberger and Steinberger, 1976; Bicsak et al., 1977). However, in vivo administration of hCG and LH also increases immunoactive inhibin levels (McLachlan et al., 1988; Drummond et al., 1989). We have demonstrated that the presence of Leydig cells is necessary for this effect (Drummond et al. 1989) and proposed that this action of LH was achieved by stimulation of Leydig cell products
Address for correspondence: of Anatomy, Monash University, 0303-7207/89/$03.50
G.P. Risbridger, Department Melbourne, Vie., Australia.
0 1989 Elsevier Scientific
Publishers
Ireland,
which in turn led to an increase in Sertoli cell inhibin production. Alternatively, LH/hCG may directly stimulate the Leydig cells to produce immunoactive i&bin. This study examines the production of inhibin by purified adult rat Leydig cells in vitro as measured by Northern blot analysis of a-subunit mRNA and by radioimmunoassay and in vitro bioassay. Materials and methods Percoll-purified Leydig cells were prepared as previously described (Risbridger et al., 1986) from adult male Sprague-Dawley rats and cultured for 20 h at 32°C in 250 ~1 DMEM (Gibco, U.S.A.) +O.l% BSA (Sigma, U.S.A.). The percentage of Ltd.
120
Leydig cells in the preparation was 89.1 k 4.3% as determined by staining for the enzyme 3/3-hydroxysteroid dehydrogenase. The cells were stimulated by addition of hormones, LH or FSH (NIADDK rLH-I-6 and rFSH-I-6). The media were collected and assayed for inhibin by radioimmunoassay as previously described using a rat ovarian extract (ROVE A) as standard (Robertson et al., 1988). In order to measure bioactive inhibin, pools of media were obtained in which Leydig cells were cultured with or without rLH or rFSH (40 ng/ml). The media were filtered on PD-10 columns (Pharmacia, Sweden) to remove testosterone, and inhibin was measured by in vitro bioassay and immunoassay (Scott et al., 1980; Robertson et al., 1988). Testosterone levels in pools of culture media from basal, FSH- or LH-stimulated Leydig cells were reduced from 45.7, 46.8 and 860 ng/ml to 0.8, 0.5 and 2.5 ng/ml respectively following gel filtration. Pituitary cell content of FSH was not significantly altered in the presence of 10 nM (i.e. 2.8 ng/ml) testosterone (18.3 k 1.3 ng FSH) compared to control (16.0 k 1.0 ng FSH, mean _t SD). All experiments were repeated at least 3 times. Total RNA was isolated from tissues or Leydig cell preparations and Northern blotting performed as previously described (Clements et al., 1988). Seminiferous tubule segments were manually dissected from adult rat testes (Gonzales et al., 1988). Interstitial or intertubular cells were prepared by collagenase dispersion of adult rat testis prior to purification of Leydig cells on gradients of Percoll (Risbridger et al., 1986). Immature female rats were treated with 20 IU PMSG followed by 20 IU hCG 48 h later and the ovaries removed 12 h after the last injection. Hybridization was performed with a genomic a-subunit inhibin cRNA probe (Albiston et al., 1989). Rehybridization with a rat 18s ribosomal RNA oligonucleotide probe (30mer) (Chan et al., 1984) was used to assess the amount and integrity of total RNA loaded onto each lane of the gel. Results Detection of mRNA. a-Subunit inhibin mRNA of equivalent size to that previously reported (Albiston et al., 1989) was demonstrated in ovary,
t2
__
I-
ctl Q 0 -1
0
-1
-2
____---
-_--------
P
I
1
I __-_-_---_-------_----
0.01)
0.15
INHISIN L
I
0
12
TUBULE
I
0.30
CULTURE
SO MEDIA
20
MEDIA
I
40
(PII
-4
0.00
STANDARD I
26
I
10
LC I
I
,h,
I
6
1.20 (U)
, 100 (j~l)
Fig. 1. Logit log-dose transformed binding curves of rat ovarian extract (ROVE A) reference preparation, seminiferous tubule culture media and Leydig cell (LC) culture media in the inhibin radioimmunoassay.
testis and seminiferous tubule mRNA (Fig. 3). A hybridization band of equivalent size was observed in two separate Leydig cell mRNA preparations; the difference in signal intensity was due to the difference in the amount of mRNA loaded onto the gel as indicated by the 18s ribosomal RNA hybridisation profile. There was no detectable signal in the crude interstitial cell RNA preparations. Inhibin production. Immunoactive inhibin was released by purified adult rat Leydig cells in vitro and detected in culture medium after 20 h. The culture media diluted in parallel to seminiferous tubule culture media and a rat overian inhibin standard preparation in the radioimmunoassay (Fig. 1). There was a linear relationship between
TABLE EFFECT LEYDIG
1 OF INCREASING NUMBER OF PURIFIED CELLS ON IMMUNOACTIVE INHIBIN LEVELS
Mean + SD, n = 3.
Cell number ( X 10 ‘/well) 1 2 4
I&bin
(U/ml)
Control
+LH
0.88 f 0.26 1.60 * 0.08 3.39 + 0.28
24450.31 5.s4*0.57 9.97 + 0.98
121
0
L 4\’ 0
f 0.S
HORYONE
I
I
I
CONCENTNATtON
JO
=
*
a
tnolh
Fig. 2. The effect of increasing doses of rLH (open symbols) or rFSH (closed symbols) on immunoactive inhibin (triangles) and testosterone (circles) production by purified Leydig cells. Each point is mean i SD, n = 4 replicate wells.
the number of Leydig cells in culture and the production of immunoactive inhibin in either the absence or presence of rLH (Table 1). Increasing doses of rat LH stimulated inhibin production by Leydig cells in a dose-related manner (Fig. 2) resulting in a 3-fold increase in inhibin. The dose
123456
Fig. 3. Northern blot of 32P-labelled a-inhibin cRNA (I) or 18s ribosomal RNA oligonucleotide (II) probes hybridised with 25 pg total RNA from PMSG/hCG stimulated ovary (lane I), seminiferous tubules (lane 2), purified Leydig cell preparation 1 (lane 3), purified Leydig cell preparation 2 (lane 4), interstitial cells (lane 5), and testis from 3%day-old rats (lane 6). Autoradio~aphy was for 4 h (~-in~~rn~ and 16 h (18 S) with Kodak XAR-5 film. Size (kb) and position of the RNA markers (BRL, Gaithersburg, MD, U.S.A.) are indicated at the right of the figure.
of rLH required to half-m~mally stimulate immunoactive inhibin production was approximately 1.2 ng,/ml. Doses of rFSH (10 and 40 ng/ml) did not significantly stimulate immunoactive inhibin production. Parallel studies demonstrated a dosedependent stimulation of testosterone following LH stimulation (Fig. 2). Bioactive inhibin levels in basal Leydig cell culture media were 2.13 f 0.98 U/ml and no significant change ( p > 0.05) occurred following stimulation with either LH (1.24 + 0.66) or FSH (2.42 f 1.55) (mean f SD, n = 4 experiments). Discussion
The detection of mRNA for the a-subunit of inhibin in isolated Leydig cell preparations and the presence of inhibin bioactivity and immunoactivity in media from unstimulated cultures of purified adult rat Leydig cell indicate that these cells are a source of inhibin. The definitive detection of inhibin bioactivity indicates that the i~unoacti~ty measured in the basal state is due to heterodime~c (alp) inhibin and does not represent cross-reaction of the assay with the recently isolated a-subunit product Pro-a, (Robertson et al., 1989; Sugino et al., 1989). Furthermore the ratio of bioactive : immunoactive inhibin levels in basal Leydig cell culture media was approximately 2.86 which is similar to the B: I ratio (1.82) that we have previously reported for inhibin in unstimulated Sertoli cell culture media (Risbridger et al., 1989). However, a similar definitive statement cannot be made for the dose-dependent increase of i~unoactive inhibin seen after LH stimulation as no corresponding rise in bioactivity was found. This observation may indicate that LH only stimulates cu-subunit secretion. Alternatively, Lee et al. (1989) have shown that a tumour-derived Leydig cell line produced activin and contained inhibin &subunit mRNA, and it is possible that LH stimulates Leydig cell activin production. However, it is unlikely that activin is the sole product of the Leydig cells following LH stimulation as bioactive inhibin levels were not reduced to undetectable values. Activin is not detected in our immunoassay but has antagonistic actions in the bioassay, so that any LH-stimulated activin production would obscure or reduce the
122
biopotency of co-secreted inhibin as we have observed in this study. Further studies are clearly required to determine the reasons for the failure of bioactive inhibin secretion to rise in parallel with immunoactive secretion following LH stimulation. Nevertheless we conclude that our data demonstrate that Leydig cells produce bioactive and immunoactive inhibin and express a-subunit inhibin mRNA. These results also demonstrate that the Leydig cells produce immunoactive inhibin which is stimulated by LH, whereas Sertoli cell inhibin production is controlled by FSH. The differential site of production of inhibin explains previous observations that serum immunoactive inhibin levels rise following hCG administration to rats (McLachlan et al., 1988; Drummond et al., 1989). These findings are consistent with the observation that patients with Klinefelter’s syndrome have high levels of serum inhibin associated with increased LH levels, yet the seminiferous epithelium is virtually devoid of Sertoli cells (de Kretser et al., 1989). The relative contributions of the Sertoli and Leydig cells to maintaining serum inhibin levels under normal conditions are unknown. We have demonstrated that the administration of the cytotoxic agent EDS, which specifically destroys Leydig cells, does not result in a significant decrease in serum immunoactive inhibin levels (de Kretser et al., 1989), suggesting that the Leydig cells are not the major source of testicular inhibin under normal conditions. This is not supported by numerous demonstrations that damage to the Sertoli cells by cryptorchidism, heat treatment or efferent duct ligation all result in a significant decrease in testicular inhibin levels. The findings presented herein clearly demonstrate that a reap-
praisal is required of our current production of inhibin and related testis.
concept proteins
of the by the
References Albiston, A., Lock, P, Burger, H.G. and Krozowski, Z.S. (1989) Proceedings of 11th Annual Conference on the Organisation and Expression of the Genome, Lome, Australia, Abstract 001-003. Bicsak, T.A., Vale, W., Vaughan, J., Tucker, M., Cappel, S. and Hsueh, A.J. (1987) Mol. Cell. Endocrinol. 49, 211-217. Chan, Y.-L., Guttell, R.R., Noller, H.F. and Wool, I.G.F. (1984) J. Biol. Sci. 259, 224-230. Clements, J.A., Matheson, B.A., Brady, J.M., Wines, D.R.. MacDonald, J.R. and Funder, J.W. (1988) J. Biol. Chem. 263, 16132-16137. de Kretser, D.M., McLachlan, R.I., Robertson, D.M. and Burger, H.G. (1989) J. Endocrinol. 120, 517-523. Drummond, A., Risbridger, G.P. and de Kretser, D.M. (1989) Endocrinology 125 (in press). Gonzales, G., Risbridger, G.P. and de Kretser, D.M. (1988) Mol. Cell. Endocrinol. 59, 179-185. Lee, W., Mason, A.J., Schwall, R., Szonyi, E. and Mather, J.P. (1988) Science 243, 396-398. McLachlan, R.I., Matsumoto, A.M., Burger, H.G., de Kretser, D.M. and Bremner, W.J. (1988) J. Clin. Invest. 82, l-5. Risbridger, G.P., Jenkin, G. and de Kretser, D.M. (1986) J. Reprod. Fertil. 77, 239-245. Risbridger, G.P., Hancock, A., Robertson, D.M., Hodgson, Y. and de Kretser, D.M. (1989) Mol. Cell. Endocrinol. 67 (in press). Robertson, D.M., Hayward, S., Irby, D.C., Jacobsen, J.V., Clarke, L.J., McLachlan, R.I. and de Kretser, D.M. (1988) Mol. Cell. Endocrinol. 58, l-8. Robertson, D.M., Giacometti, E., Foulds, L.M., Lahnstein, J., Goss, N., Heam, M.T.W. and de Kretser, D.M. (1989) Endocrinology (in press). Scott, RX, Burger, H.G. and Quigg, H. (1980) Endocrinology 107, 1536-1542. Steinberger, A. and Steinberger, E. (1976) Endocrinology 99, 918-921. Sugino, K., Nakamira, T., Takio, K., Titani, K., Miyamoto, K., Hasegawa, Y., Igarashi, M. and Sugino, H. (1989) B&hem. Biophys. Res. Commun. 159, 1323-1329.
MOLCEL
119
02153
Rapid Paper
Immuno- and bioactive inhibin and inhibin a-subunit Leydig cell cultures G.P. Risbridger
expression in rat
‘, J. Clements 2, D.M. Robertson I, A.E. Drummond H.G. Burger * and D.M. de Kretser ’
I, J. Muir I,
’ Department of Anatomy,Monash University, Melbourne, Victoria, Australia, and ’ Medical Research Centre, Prince Henry’s Hospital, Melbourne, Victoria, Australia (Accepted
Key words: Leydig
cell; Inhibin;
10 July 1989)
(Rat)
Summary The rise in serum immunoactive inhibin levels in male rats following hCG stimulation raised the possibility that Leydig cells may produce inhibin. This study therefore evaluated whether Percoll-purified Leydig cells from adult male rats synthesized and secreted inhibin in vitro as measured by Northern blot analysis, radioimmunoassay and in vitro bioassay. Northern blot analysis demonstrated the presence of cx-inhibin subunit mRNA in the Leydig cell and inhibin bioactivity was detected in Leydig cell culture media. Levels of immunoactive inhibin increased in culture over 20 h and were directly dependent on the number of Leydig cells in culture. rLH (NIADDK-rLH-I-6) and not rFSH (NIADDK-rFSH-I-6) stimulated immunoactive inhibin levels in a dose-depedent manner. This study demonstrates that Leydig cells express mRNA for the a-subunit of inhibin and produce inhibin which is biologically and immunologically active prompting a re-evaluation of our concepts of testicular inhibin production.
Introduction Inhibin is a glycoprotein hormone that is produced by the Sertoli cells and regulated by FSH (Steinberger and Steinberger, 1976; Bicsak et al., 1977). However, in vivo administration of hCG and LH also increases immunoactive inhibin levels (McLachlan et al., 1988; Drummond et al., 1989). We have demonstrated that the presence of Leydig cells is necessary for this effect (Drummond et al. 1989) and proposed that this action of LH was achieved by stimulation of Leydig cell products
Address for correspondence: of Anatomy, Monash University, 0303-7207/89/$03.50
G.P. Risbridger, Department Melbourne, Vie., Australia.
0 1989 Elsevier Scientific
Publishers
Ireland,
which in turn led to an increase in Sertoli cell inhibin production. Alternatively, LH/hCG may directly stimulate the Leydig cells to produce immunoactive i&bin. This study examines the production of inhibin by purified adult rat Leydig cells in vitro as measured by Northern blot analysis of a-subunit mRNA and by radioimmunoassay and in vitro bioassay. Materials and methods Percoll-purified Leydig cells were prepared as previously described (Risbridger et al., 1986) from adult male Sprague-Dawley rats and cultured for 20 h at 32°C in 250 ~1 DMEM (Gibco, U.S.A.) +O.l% BSA (Sigma, U.S.A.). The percentage of Ltd.
120
Leydig cells in the preparation was 89.1 k 4.3% as determined by staining for the enzyme 3/3-hydroxysteroid dehydrogenase. The cells were stimulated by addition of hormones, LH or FSH (NIADDK rLH-I-6 and rFSH-I-6). The media were collected and assayed for inhibin by radioimmunoassay as previously described using a rat ovarian extract (ROVE A) as standard (Robertson et al., 1988). In order to measure bioactive inhibin, pools of media were obtained in which Leydig cells were cultured with or without rLH or rFSH (40 ng/ml). The media were filtered on PD-10 columns (Pharmacia, Sweden) to remove testosterone, and inhibin was measured by in vitro bioassay and immunoassay (Scott et al., 1980; Robertson et al., 1988). Testosterone levels in pools of culture media from basal, FSH- or LH-stimulated Leydig cells were reduced from 45.7, 46.8 and 860 ng/ml to 0.8, 0.5 and 2.5 ng/ml respectively following gel filtration. Pituitary cell content of FSH was not significantly altered in the presence of 10 nM (i.e. 2.8 ng/ml) testosterone (18.3 k 1.3 ng FSH) compared to control (16.0 k 1.0 ng FSH, mean _t SD). All experiments were repeated at least 3 times. Total RNA was isolated from tissues or Leydig cell preparations and Northern blotting performed as previously described (Clements et al., 1988). Seminiferous tubule segments were manually dissected from adult rat testes (Gonzales et al., 1988). Interstitial or intertubular cells were prepared by collagenase dispersion of adult rat testis prior to purification of Leydig cells on gradients of Percoll (Risbridger et al., 1986). Immature female rats were treated with 20 IU PMSG followed by 20 IU hCG 48 h later and the ovaries removed 12 h after the last injection. Hybridization was performed with a genomic a-subunit inhibin cRNA probe (Albiston et al., 1989). Rehybridization with a rat 18s ribosomal RNA oligonucleotide probe (30mer) (Chan et al., 1984) was used to assess the amount and integrity of total RNA loaded onto each lane of the gel. Results Detection of mRNA. a-Subunit inhibin mRNA of equivalent size to that previously reported (Albiston et al., 1989) was demonstrated in ovary,
t2
__
I-
ctl Q 0 -1
0
-1
-2
____---
-_--------
P
I
1
I __-_-_---_-------_----
0.01)
0.15
INHISIN L
I
0
12
TUBULE
I
0.30
CULTURE
SO MEDIA
20
MEDIA
I
40
(PII
-4
0.00
STANDARD I
26
I
10
LC I
I
,h,
I
6
1.20 (U)
, 100 (j~l)
Fig. 1. Logit log-dose transformed binding curves of rat ovarian extract (ROVE A) reference preparation, seminiferous tubule culture media and Leydig cell (LC) culture media in the inhibin radioimmunoassay.
testis and seminiferous tubule mRNA (Fig. 3). A hybridization band of equivalent size was observed in two separate Leydig cell mRNA preparations; the difference in signal intensity was due to the difference in the amount of mRNA loaded onto the gel as indicated by the 18s ribosomal RNA hybridisation profile. There was no detectable signal in the crude interstitial cell RNA preparations. Inhibin production. Immunoactive inhibin was released by purified adult rat Leydig cells in vitro and detected in culture medium after 20 h. The culture media diluted in parallel to seminiferous tubule culture media and a rat overian inhibin standard preparation in the radioimmunoassay (Fig. 1). There was a linear relationship between
TABLE EFFECT LEYDIG
1 OF INCREASING NUMBER OF PURIFIED CELLS ON IMMUNOACTIVE INHIBIN LEVELS
Mean + SD, n = 3.
Cell number ( X 10 ‘/well) 1 2 4
I&bin
(U/ml)
Control
+LH
0.88 f 0.26 1.60 * 0.08 3.39 + 0.28
24450.31 5.s4*0.57 9.97 + 0.98
121
0
L 4\’ 0
f 0.S
HORYONE
I
I
I
CONCENTNATtON
JO
=
*
a
tnolh
Fig. 2. The effect of increasing doses of rLH (open symbols) or rFSH (closed symbols) on immunoactive inhibin (triangles) and testosterone (circles) production by purified Leydig cells. Each point is mean i SD, n = 4 replicate wells.
the number of Leydig cells in culture and the production of immunoactive inhibin in either the absence or presence of rLH (Table 1). Increasing doses of rat LH stimulated inhibin production by Leydig cells in a dose-related manner (Fig. 2) resulting in a 3-fold increase in inhibin. The dose
123456
Fig. 3. Northern blot of 32P-labelled a-inhibin cRNA (I) or 18s ribosomal RNA oligonucleotide (II) probes hybridised with 25 pg total RNA from PMSG/hCG stimulated ovary (lane I), seminiferous tubules (lane 2), purified Leydig cell preparation 1 (lane 3), purified Leydig cell preparation 2 (lane 4), interstitial cells (lane 5), and testis from 3%day-old rats (lane 6). Autoradio~aphy was for 4 h (~-in~~rn~ and 16 h (18 S) with Kodak XAR-5 film. Size (kb) and position of the RNA markers (BRL, Gaithersburg, MD, U.S.A.) are indicated at the right of the figure.
of rLH required to half-m~mally stimulate immunoactive inhibin production was approximately 1.2 ng,/ml. Doses of rFSH (10 and 40 ng/ml) did not significantly stimulate immunoactive inhibin production. Parallel studies demonstrated a dosedependent stimulation of testosterone following LH stimulation (Fig. 2). Bioactive inhibin levels in basal Leydig cell culture media were 2.13 f 0.98 U/ml and no significant change ( p > 0.05) occurred following stimulation with either LH (1.24 + 0.66) or FSH (2.42 f 1.55) (mean f SD, n = 4 experiments). Discussion
The detection of mRNA for the a-subunit of inhibin in isolated Leydig cell preparations and the presence of inhibin bioactivity and immunoactivity in media from unstimulated cultures of purified adult rat Leydig cell indicate that these cells are a source of inhibin. The definitive detection of inhibin bioactivity indicates that the i~unoacti~ty measured in the basal state is due to heterodime~c (alp) inhibin and does not represent cross-reaction of the assay with the recently isolated a-subunit product Pro-a, (Robertson et al., 1989; Sugino et al., 1989). Furthermore the ratio of bioactive : immunoactive inhibin levels in basal Leydig cell culture media was approximately 2.86 which is similar to the B: I ratio (1.82) that we have previously reported for inhibin in unstimulated Sertoli cell culture media (Risbridger et al., 1989). However, a similar definitive statement cannot be made for the dose-dependent increase of i~unoactive inhibin seen after LH stimulation as no corresponding rise in bioactivity was found. This observation may indicate that LH only stimulates cu-subunit secretion. Alternatively, Lee et al. (1989) have shown that a tumour-derived Leydig cell line produced activin and contained inhibin &subunit mRNA, and it is possible that LH stimulates Leydig cell activin production. However, it is unlikely that activin is the sole product of the Leydig cells following LH stimulation as bioactive inhibin levels were not reduced to undetectable values. Activin is not detected in our immunoassay but has antagonistic actions in the bioassay, so that any LH-stimulated activin production would obscure or reduce the
122
biopotency of co-secreted inhibin as we have observed in this study. Further studies are clearly required to determine the reasons for the failure of bioactive inhibin secretion to rise in parallel with immunoactive secretion following LH stimulation. Nevertheless we conclude that our data demonstrate that Leydig cells produce bioactive and immunoactive inhibin and express a-subunit inhibin mRNA. These results also demonstrate that the Leydig cells produce immunoactive inhibin which is stimulated by LH, whereas Sertoli cell inhibin production is controlled by FSH. The differential site of production of inhibin explains previous observations that serum immunoactive inhibin levels rise following hCG administration to rats (McLachlan et al., 1988; Drummond et al., 1989). These findings are consistent with the observation that patients with Klinefelter’s syndrome have high levels of serum inhibin associated with increased LH levels, yet the seminiferous epithelium is virtually devoid of Sertoli cells (de Kretser et al., 1989). The relative contributions of the Sertoli and Leydig cells to maintaining serum inhibin levels under normal conditions are unknown. We have demonstrated that the administration of the cytotoxic agent EDS, which specifically destroys Leydig cells, does not result in a significant decrease in serum immunoactive inhibin levels (de Kretser et al., 1989), suggesting that the Leydig cells are not the major source of testicular inhibin under normal conditions. This is not supported by numerous demonstrations that damage to the Sertoli cells by cryptorchidism, heat treatment or efferent duct ligation all result in a significant decrease in testicular inhibin levels. The findings presented herein clearly demonstrate that a reap-
praisal is required of our current production of inhibin and related testis.
concept proteins
of the by the
References Albiston, A., Lock, P, Burger, H.G. and Krozowski, Z.S. (1989) Proceedings of 11th Annual Conference on the Organisation and Expression of the Genome, Lome, Australia, Abstract 001-003. Bicsak, T.A., Vale, W., Vaughan, J., Tucker, M., Cappel, S. and Hsueh, A.J. (1987) Mol. Cell. Endocrinol. 49, 211-217. Chan, Y.-L., Guttell, R.R., Noller, H.F. and Wool, I.G.F. (1984) J. Biol. Sci. 259, 224-230. Clements, J.A., Matheson, B.A., Brady, J.M., Wines, D.R.. MacDonald, J.R. and Funder, J.W. (1988) J. Biol. Chem. 263, 16132-16137. de Kretser, D.M., McLachlan, R.I., Robertson, D.M. and Burger, H.G. (1989) J. Endocrinol. 120, 517-523. Drummond, A., Risbridger, G.P. and de Kretser, D.M. (1989) Endocrinology 125 (in press). Gonzales, G., Risbridger, G.P. and de Kretser, D.M. (1988) Mol. Cell. Endocrinol. 59, 179-185. Lee, W., Mason, A.J., Schwall, R., Szonyi, E. and Mather, J.P. (1988) Science 243, 396-398. McLachlan, R.I., Matsumoto, A.M., Burger, H.G., de Kretser, D.M. and Bremner, W.J. (1988) J. Clin. Invest. 82, l-5. Risbridger, G.P., Jenkin, G. and de Kretser, D.M. (1986) J. Reprod. Fertil. 77, 239-245. Risbridger, G.P., Hancock, A., Robertson, D.M., Hodgson, Y. and de Kretser, D.M. (1989) Mol. Cell. Endocrinol. 67 (in press). Robertson, D.M., Hayward, S., Irby, D.C., Jacobsen, J.V., Clarke, L.J., McLachlan, R.I. and de Kretser, D.M. (1988) Mol. Cell. Endocrinol. 58, l-8. Robertson, D.M., Giacometti, E., Foulds, L.M., Lahnstein, J., Goss, N., Heam, M.T.W. and de Kretser, D.M. (1989) Endocrinology (in press). Scott, RX, Burger, H.G. and Quigg, H. (1980) Endocrinology 107, 1536-1542. Steinberger, A. and Steinberger, E. (1976) Endocrinology 99, 918-921. Sugino, K., Nakamira, T., Takio, K., Titani, K., Miyamoto, K., Hasegawa, Y., Igarashi, M. and Sugino, H. (1989) B&hem. Biophys. Res. Commun. 159, 1323-1329.