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Effect of diverse mammalian and nonmammalian gonadotropins on isolated rat Leydig cells
GENERAL
AND
COMPARATIVE
32,488-494
ENDOCRINOLOGY
(1977)
Effect of Diverse Mammalian and Nonmammalian Gonadotropins on Isolated Rat Leydig Cells SUSAN WALKER FARMER,* ALAN SUYAMA,*? AND HAROLD PAPKOFF*? *Hormone
Research University
Laboratory of California,
and tReproductive San Francisco,
Endocrinology California 94143
Center,
Accepted March 7, 1977 The isolated rat Leydig cell assay was employed to study species specificity in biological action. Eight mammalian luteinizing hormones (LHs), human chorionic gonadotropin, and pregnant mare serum gonadotropin stimulated testosterone production in this assay with a 250-fold potency range. A high potency correlated with a high carbohydrate content. Comparison of the potencies of these hormones in the Leydig assay, the ovarian ascorbic acid depletion assay, and the Xenopus ovulation assay showed the results of the two rat assays to be the most similar, demonstrating that the receptor as well as the hormone play a role in determining species specificity. The LH specificity of the assay was reexamined and follicle-stimulating hormone (FSH) was found to have a small intrinsic activity. Nonmammalian LHs, which generally possess little or no activity in in vivo mammalian assays, were able to stimulate rat Leydig cells, at 1000x mammalian LH doses. LH specificity was retained with nonmammalian LHs but the potency differences between nonmammalian LHs and FSHs were much less than between mammalian LHs and FSHs.
Studies from this laboratory (Moyle and Ramachandran, 1973; Ramachandran and Sairam, 1975) and other studies (Dufau et al., 1974, 1976), have demonstrated that isolated Leydig cell preparations from rat testes produce testosterone in response to stimulation from several species of mammalian LH, and that this response can be used as a very sensitive and specific in vitro bioassay for LH. We have employed this assay to study species specificity in gonadotropic activity, particularly the ability of nonmammalian gonadotropins to stimulate mammalian tissues. In addition to hCG’ and PMSG, eight species of mammalian LH and five nonmammalian LHs and FSHs were tested. Further, the r Abbreviations used: LH, luteinizing hormone; hLH, human luteinizing hormone; FSH, folliclestimulating hormone; TSH, thyroid-stimulating hormone; hCG, human chorionic gonadotropin; PMSG, pregnant mare serum gonadotropin; AS, antiserum; OAAD assay, ovarian ascorbic acid depletion assay.
specificity of the assay for LH was reexamined. It was found that ovine FSH had low but intrinsic activity in this system. MATERIALS
AND METHODS
Hormone preparations. Most of the hormones employed in this study were prepared in this laboratory, with the exception of the NIH-LH-S18, rat LH (RLH-I, NIAMMD), and hCG (gifts of the NIH Hormone Distribution Program). Bovine TSH was a gift from Dr. John G. Pierce, UCLA, Los Angeles, California. The preparation of PMSG (Schams and Papkoff, 1972) and some of the mammalian LHs have been described: ovine (Papkoff et al., 1965); bovine (Papkoff and Can, 1970). The other LHs, human, horse, pig, dog, and rabbit, were purified by methods very similar to those employed with ovine LH. Ovine FSH was prepared as described by Papkoff (1973). The preparation of the nonmammalian gonadotropins has been described in several publications (Farmer et al., 1975; Papkoff et al., 1976a,b; Licht et al., 1976a). Leydig cell assay. Leydig cell suspensions were obtained essentially as described previously (Ramachandran and Sairam, 1975) from 140- to 160-g Sprague-Dawley rats, except that a slightly shorter incubation time of 30 min with collagenase was em-
488 Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 00166430
EFFECT
OF GGNADOTROPINS
ployed. Leydig cell suspensions (0.250 ml, 0.1-10 x IOr’ cells/ml) were incubated in duplicate in plastic tubes, with hormone preparations in 0.05 ml of saline, for 2 hr at 37” in an atmosphere of 95% 0,:5% CO,. Incubation was terminated by the addition of 1.0 ml of cold 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% bovine serum albumin. The testosterone content of suitable aliquots was directly measured by radioimmunoassay. This was performed as described previously (Ramachandran and Sairam, 1975), except that the sample aliquots were incubated for approximately 16 hr at 4” after addition of [3H]testosterone and antiserum, and PCS scintillant (Amersham Searle) was used. An ahquot of the cell suspension was examined under a light microscope, and the large Leydig cells were counted with the aid of a hemacytometer. The results are expressed as nanograms of testosterone per IO6 cells, and the hormone concentrations are expressed as nanograms or micrograms per tube (0.3 ml). Parallel-line assay statistics for linearity, precision, parallelism, and relative potency estimates were computed according to the method of Finney (1964), and the results from replicate assays were averaged. Indexes of precision (A) for these assays ranged from 0.03 to 0.22.
RESULTS
Specljkity The isolated rat Leydig cell system has been shown to be a sensitive and specific assay for LH (Moyle and Ramachandran,
ON LEYDIG
489
CELLS
1975; Dufau et al., 1976). In the latter study, human FSH was found to have low activity which was blocked by preincubation in the presence of antiserum to hCG or to hLH. In our study, however, a doseresponse curve was obtained with ovine FSH which was parallel to that for ovine LH, but was displaced toward higher concentrations (2000x). Studies with rabbit anti-ovine LH serum were conducted to determine if the FSH response was intrinsic or due to LH contamination. Results of these studies are presented in Fig. 1. It is seen that the addition of LH antiserum to the LH (200-500 /.q of LWml of AS) completely inhibited the production of testosterone (Fig. 1A). Addition of normal rabbit serum did not affect the LH response. When ovine FSH was assayed with LH antiserum (1 and 5 mg of FSWml of AS), there was a partial reduction (33%) in the response when compared to FSH alone (Fig. 1B). Thus, while there appears to be a trace of LH in the FSH preparation, the response to the antiserum-treated FSH was dose dependent and significantly higher than the controls in each case, and it prob-
r
- 60 ’ t -4of
:: t -20 @ @
FIG. I. The effect of LH antiserum dig cells by LH (A) and FSH (R).
on the stimulation
of testosterone
production
in isolated rat Ley-
490
FARMER,
SUYAMA,
ably represents a small but intrinsic activity of the FSH.’ Mammalian
LHs
The stimulation of testosterone production by isolated Leydig cells with highly purified LHs from eight mammalian species, as well as with hCG and PMSG, is shown in Fig. 2. The data in the left panel are from a single experiment, and those in the right panel are from four experiments which showed an identical response for ovine LH [G3-222B (Papkoff et al., 1965)l. This ovine LH was run in all experiments as a standard; Fig. 2 (right panel) also includes the response of another standard, NIH-LH-S18. All of the LHs gave parallel log dose-related responses, with the same maxima when high enough doses were assayed. Averaged potency estimates for these hormones are listed in Table 1. There was a very wide range of potencies (250fold) for the various LHs, all of which are of comparable purity. Species variation can 2 Bovine TSH was also found to stimulate testosterone production in a manner comparable to ovine LH at a high concentration (500x ovine LH), but the specificity of this response was not checked with antiserum.
AND
PAPKOFF
also be seen with the same preparations assayed in both the ovarian ascorbic acid depletion (OAAD) assay (fivefold range) and the Xenopus ovulation assay (lOO-fold range) [Table 1, Licht and Papkoff (1976)l. There was no consistent relationship in potency rank among the species, but the results of the two mammalian assays were the most similar. Nonmammalian
Gonaa’otropins
LHs from nonmammalian species also stimulated testosterone production in rat Leydig cells in a manner identical to that of ovine LH, but at approximately a lOOO-fold greater dose level. These results are shown in Fig. 3, with LHs from the turkey, alligator, snapping turtle, sea turtle, and bullfrog. The potencies of these various LHs were quite similar; there was only a six-fold difference, compared with the 250fold potency range observed with LHs from mammalian species (Table 2). FSHs from these same nonmammalian species were assayed at one or two dose levels. They were all active, at higher dose levels than the respective LHs, but were much closer in potency to their LHs than were mammalian FSHs. These data are summarized in
iQo=” 60. 2 % \ 60t H 40!! E B m-
HORMONE
FIG. 2. Stimulation d testosterone production from eight mammalian species.
CONCENTRATION
(ng)
in isolated rat Leydig cells by hCG, PMSG, and LHs
EFFECT
OF GONADOTROPINS TABLE
BIOLOGICAL
ACTIVITIES AND
Species hCG Human LH Horse LH PMSG Sheep LH Ox LH Pig LH Rat LH Rabbit LH Dog LH
AND
SEVERAL
491
CELLS
I
CARBOHYDRATE
CONTENT
MAMMALIAN
Total carbohydrate content a 34 28 24 45 16 13 14 15 ND ND
ON LEYDIG
OF
hCG, PMSG,
LHs Biological
Leydig’
25.3 (17.3-35.2) 9.7 (6.8-14.0) 6.3 (2.7-l 1.O) 2.6 (I .9-3.8) 1.6 (1.1-2.6) 0.9 (0.5-I .5) 0.4 (0.2-0.6) 0.4 (0.2-0.7) 0.2 (0.1-0.5) 0. I (0.04-0.2)
potencies
OAADd NDe 4.1 3.0 1.5 2.8 1.0 1.2 1.0 0.8 ND
*
Xenopus
ND 0.05 0.6 ND 2.8 1.3 0.1 <0.25 0.025 ND
a Grams/l00 g of protein. * All potency estimates are x NIH-LH-Sl. ’ Averaged potency values; 95% confidence limits are in parentheses. d Taken from Licht and Papkoff (1976). e ND, not determined.
Table 3, where the potencies of the various species of FSH are given in terms of the LH from the same species. Thus, while ovine FSH is active only at levels 2300x greater than ovine LH, snapping turtle FSH is equally active at levels only 17x greater than snapping turtle LH. DISCUSSION
The data presented here show the response of isolated Leydig cells to a wide variety of mammalian and nonmammalian LHs. All of these LHs were prepared by similar methodology and are of comparable purity, facilitating comparisons. The range of species studied includes at least one representative species from each tetrapod class and representatives of six different mammalian orders. Similar results with some of these hormones have been reported previously by Ramachandran and Sairam (1975) and Dufau et al. (1976). The fact that all of these species of LH, as well as hCG and PMSG, were able to stimulate testosterone production in rat Leydig cells attests to the basic similarity of these LHs and the wide applicability of the assay.
The insensitivity of one species to the hormones derived from a different species is termed species specificity. It is known that the structures of both the hormone and the target cell receptor play a role in determining species specificity. In our study, the use of an in vitro assay eliminated the problem of differences in the metabolic clearance of the hormones, and thus the variation in potency observed among the LH species (250-fold) must relate to structural differences which affect their interaction with the target tissue. The primary structures of hCG and five of the LHs employed are known and the differences in amino acid sequences among these species could account for the potency differences observed. In addition, in our studies a high content of carbohydrate appeared to correlate well with high activity (Table 1). The relationship of carbohydrate and hCG potency has been directly studied by Moyle et al. (1975). They observed that the removal of sugar residues from hCG caused a reduction in potency in the rat Leydig cell assay while parallelism and the response maxima were not affected. The response of PMSG
d
492
FARMER,
P
SUYAMA,
AND
PAPKOFF
20.
0
I WI
lb,
I
HORMONE
CONCENTRATION
FIG. 3. Stimulation of testosterone production in isolated rat Leydig cells by sheep LH and LHs from five nonmammalian species. Note different concentration ranges employed for the mammalian (nanogram) and nonmammalian (microgram) LHs.
TABLE
2
BIOLOGICAL ACTIVITIES OF NONMAMMALIAN LEYDIG CELL ASSAY AND THE Xenopus
LHs MEASURED OVULATION
Bioloeical
Turkey Alligator Snapping turtle Sea turtle Bullfrog
2.6 I.1 I. I 0.5 0.8
a Taken from Farmer et al., TABLE
1975; Licht er a/., 3
BIOLOGICAL POTENCIES OF SEVERAL SPECIESOF FSH MEASURED AGAINST THE SAME SPECIES OF LH IN THE RAT LEYDIG CELL ASSAY
Sheep ox Turkey Alligator Sea turtle Snapping turtle Bullfrog
Potency (x LH of same species) 0.005 0.005 0.36 0.03 0.07 0.06 0.26
Xenopus
x NIH-LH-S
(I .8-4.2) (0.8-1.3) (0.8-I .4) (0.4-0.6) (0.5-I .2)
1976a; Papkoff et al.,
RAT
uotencies
Leydig lO-3 x NIH-LH-Sl (95% limits)
LH obtained from
FSH obtained from
IN THE ASSAY
a
1
0.3 3.3 1.8 I.0 0.3 1976a,b.
is notable because this aonadotroninI has the highest carbohydrate content but was ranked only fourth in Leydig cell potency (Table 1). This is in part due to the fact that the potency calculations employed nanogram per milliliter hormone concentrations rather than molarity, since the precise molecular weights of most of the hormones employed are not known. In addition, PMSG is known to possess both LH and FSH activities and may therefore have an amino acid sequence somewhat different from the sequences of the other LHs
EFFECT
OF GONADOTROPINS
tested, which may also account for its relative potency. Data obtained with the same LH preparations in two other bioassays, the OAAD and Xenopus ovulation, were also shown in Table 1. The potency differences observed for each hormone in the Leydig and OAAD assays are interesting in that comparisons are made for responses which were obtained in vitro versus in vivo and in male versus female and for a physiological parameter (testosterone production) versus a response of unknown significance (ascorbic acid depletion). Any of these factors could account for the LH potency differences observed between the two assays. The potency ranking, however, is similar in these two rat assays and quite different from that seen in the frog assay. This comparison supports the idea that the receptor as well as the hormone play a role in determining species differences. In the past it has proven difficult to assay nonmammalian gonadotropins in in vivo mammalian assays. With the use of sensitive in vitro assays, however, both we and Licht et al. (1976b) have been able to demonstrate an effect of nonmammalian gonadotropins on mammalian tissue, although lOOO-fold greater dose levels were necessary. The nonmammalian LHs are of purity comparable to that of the mammalian hormones , as judged by chemical criteria (Farmer et al., 1975; Papkoff et al., 1976a; Licht et al., 1976a) and by their potencies in the Xenopus ovulation assay (Table 2). Therefore, there is indeed great specificity of rat Leydig cells for mammalian hormones. Nevertheless, because of its sensitivity, this assay can be used to advantage in studies on nonmammalian hormones. LH specificity in the Leydig cell assay was retained with the nonmammalian hormones. However, the potency differences between the nonmammalian LHs and FSHs were much closer than those found with the mammalian hormones (Table 3). It is of interest that mammalian FSH and TSH give
ON LEYDIG
CELLS
493
dose-responses at the same concentrations at which nonmammalian gonadotropins are active. These data could suggest that, at these high hormone concentrations, the Leydig cell begins to respond to structural features which the glycoprotein hormones all have in common. In conclusion, our results demonstrate the usefulness of the rat Leydig cell assay for structure-function studies. In such studies, the variety of species of LH serve as natural analogs, since preparation of synthetic analogs of these large glycoproteins is as yet impossible. Our data confirmed the high specificity of the Leydig cell assay for mammalian LHs, which are active at nanogram levels, but also showed that, at microgram hormone levels, the Leydig cell recognizes more general glycoprotein features and responds to mammalian FSH and TSH and to nonmammalian LHs and FSHs. ACKNOWLEDGMENTS We thank Professor Choh Hao Li for his support of these studies and for critically reading the manuscript. This work was supported in part by a grant from the National Science Foundation, BMS 7516138, a Rockefeller Foundation Center Grant, and NIH Grants AM-6097 and HD-05722.
REFERENCES Dufau, M. L., Mendelson, C., and Catt, K. J. (1974). A highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin: Testosterone production by dispersed Leydig cells. J. Clin. Endocrinol. Metab. 39, 610-613. Dufau, M. L., Pock, R., Neubauer, A., and Catt, K. J. (1976). In vitro bioassay of LH in human serum: The rat interstitial cell testosterone (RICT) assay. J. Clin
Endocrinol.
Metab.
42, 958-969.
Farmer, S. W., Papkoff, H., and Licht, P. (1975). Purification of turkey gonadotropins. Biol. Reprod.
12, 415-422.
Finney, D. J. (1964). “Statistical Methods in Biological Assays,” pp. 99-163. Griffen, London. Licht, P., and Papkoff, H. (1976). Species specificity in the response of an in vitro amphibian (Xenopus laevis) ovulation assay to mammalian luteinizing hormones. Gen. Comp. Endocrinol. 29,552-555. Licht, P., Farmer, S. W.. and Papkoff, H. (1976a). Further studies on the chemical nature of reptilian
494
FARMER,
SUYAMA,
gonadotropins: FSH and LH in the American alligator and green sea turtle. Biol. Reprod. 14, 222-232. Licht, P., Mullet-, C. H., and Tsui, H. W. (1976b). Effects of mammalian and nonmammalian gonadotropins on andtogen production by minced rabbit testis. Biol. Reprod. 14, 194-201. Moyle, W. R., and Ramachandran, J. (1973). Effect of LH on steroidogenesis and cyclic AMP accumulation in rat Leydig cell preparations and mouse tumor Leydig cells. Endocrinology 93, 127-l 34. Moyle, W. R., Bahl, 0. P., and MBrz, L. (1975). Role of the carbohydrate of human chorionic gonadotropin in the mechanism of hormone action. J. Biol. Chem. 250, 9163-9169. Papkoff, H. (1973). Studies on the structure of ovine follicle stimulating hormone. In “Proceedings, 4th International Congress of Endocrinology, Washington.” Excerpta Medica Znt. Congr. Ser. 273, 596-600. Papkoff, H., and Can, J. (1970). Bovine interstitial cell-stimulating hormone: Purification and properties. Arch. Biochem. Biophys. 136, 522-528.
AND PAPKOFF Papkoff, H., Gospodarowicz, D., Candiotti, A., and Li, C. H. (196.5). Preparation of ovine interstitial cell-stimulating hormone in high yield. Arch. Biochem. Biophys. 111, 431-438. Papkoff, H., Farmer, S. W., and Licht, P. (1976a). Isolation and characterization of folliclestimulating hormone and luteinizing hormone and its subunits from snapping turtle (Chelydra serpentina) pituitaries. Endocrinology 98, 767-777. Papkoff, H., Farmer, S. W., and Licht, P. (1976b). Isolation and characterization of luteinizing hormone from amphibian (Rana catesbeiana) pituitaries. Life Sci. 18, 245-250. Ramachandran, J., and Sairam, M. R. (1975). The effects of interstitial cell-stimulating hormone, its subunits, and recombinations on isolated rat Leydig cells. Arch. Biochem. Biophys. 167, 294300. Schams, D., and Papkoff, H. (1972). Chemical and immunochemical studies on pregnant mare serum gonadotropin. Biochim. Biophys. Acta 263, 139148.
AND
COMPARATIVE
32,488-494
ENDOCRINOLOGY
(1977)
Effect of Diverse Mammalian and Nonmammalian Gonadotropins on Isolated Rat Leydig Cells SUSAN WALKER FARMER,* ALAN SUYAMA,*? AND HAROLD PAPKOFF*? *Hormone
Research University
Laboratory of California,
and tReproductive San Francisco,
Endocrinology California 94143
Center,
Accepted March 7, 1977 The isolated rat Leydig cell assay was employed to study species specificity in biological action. Eight mammalian luteinizing hormones (LHs), human chorionic gonadotropin, and pregnant mare serum gonadotropin stimulated testosterone production in this assay with a 250-fold potency range. A high potency correlated with a high carbohydrate content. Comparison of the potencies of these hormones in the Leydig assay, the ovarian ascorbic acid depletion assay, and the Xenopus ovulation assay showed the results of the two rat assays to be the most similar, demonstrating that the receptor as well as the hormone play a role in determining species specificity. The LH specificity of the assay was reexamined and follicle-stimulating hormone (FSH) was found to have a small intrinsic activity. Nonmammalian LHs, which generally possess little or no activity in in vivo mammalian assays, were able to stimulate rat Leydig cells, at 1000x mammalian LH doses. LH specificity was retained with nonmammalian LHs but the potency differences between nonmammalian LHs and FSHs were much less than between mammalian LHs and FSHs.
Studies from this laboratory (Moyle and Ramachandran, 1973; Ramachandran and Sairam, 1975) and other studies (Dufau et al., 1974, 1976), have demonstrated that isolated Leydig cell preparations from rat testes produce testosterone in response to stimulation from several species of mammalian LH, and that this response can be used as a very sensitive and specific in vitro bioassay for LH. We have employed this assay to study species specificity in gonadotropic activity, particularly the ability of nonmammalian gonadotropins to stimulate mammalian tissues. In addition to hCG’ and PMSG, eight species of mammalian LH and five nonmammalian LHs and FSHs were tested. Further, the r Abbreviations used: LH, luteinizing hormone; hLH, human luteinizing hormone; FSH, folliclestimulating hormone; TSH, thyroid-stimulating hormone; hCG, human chorionic gonadotropin; PMSG, pregnant mare serum gonadotropin; AS, antiserum; OAAD assay, ovarian ascorbic acid depletion assay.
specificity of the assay for LH was reexamined. It was found that ovine FSH had low but intrinsic activity in this system. MATERIALS
AND METHODS
Hormone preparations. Most of the hormones employed in this study were prepared in this laboratory, with the exception of the NIH-LH-S18, rat LH (RLH-I, NIAMMD), and hCG (gifts of the NIH Hormone Distribution Program). Bovine TSH was a gift from Dr. John G. Pierce, UCLA, Los Angeles, California. The preparation of PMSG (Schams and Papkoff, 1972) and some of the mammalian LHs have been described: ovine (Papkoff et al., 1965); bovine (Papkoff and Can, 1970). The other LHs, human, horse, pig, dog, and rabbit, were purified by methods very similar to those employed with ovine LH. Ovine FSH was prepared as described by Papkoff (1973). The preparation of the nonmammalian gonadotropins has been described in several publications (Farmer et al., 1975; Papkoff et al., 1976a,b; Licht et al., 1976a). Leydig cell assay. Leydig cell suspensions were obtained essentially as described previously (Ramachandran and Sairam, 1975) from 140- to 160-g Sprague-Dawley rats, except that a slightly shorter incubation time of 30 min with collagenase was em-
488 Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 00166430
EFFECT
OF GGNADOTROPINS
ployed. Leydig cell suspensions (0.250 ml, 0.1-10 x IOr’ cells/ml) were incubated in duplicate in plastic tubes, with hormone preparations in 0.05 ml of saline, for 2 hr at 37” in an atmosphere of 95% 0,:5% CO,. Incubation was terminated by the addition of 1.0 ml of cold 0.1 M sodium phosphate buffer, pH 7.4, containing 0.5% bovine serum albumin. The testosterone content of suitable aliquots was directly measured by radioimmunoassay. This was performed as described previously (Ramachandran and Sairam, 1975), except that the sample aliquots were incubated for approximately 16 hr at 4” after addition of [3H]testosterone and antiserum, and PCS scintillant (Amersham Searle) was used. An ahquot of the cell suspension was examined under a light microscope, and the large Leydig cells were counted with the aid of a hemacytometer. The results are expressed as nanograms of testosterone per IO6 cells, and the hormone concentrations are expressed as nanograms or micrograms per tube (0.3 ml). Parallel-line assay statistics for linearity, precision, parallelism, and relative potency estimates were computed according to the method of Finney (1964), and the results from replicate assays were averaged. Indexes of precision (A) for these assays ranged from 0.03 to 0.22.
RESULTS
Specljkity The isolated rat Leydig cell system has been shown to be a sensitive and specific assay for LH (Moyle and Ramachandran,
ON LEYDIG
489
CELLS
1975; Dufau et al., 1976). In the latter study, human FSH was found to have low activity which was blocked by preincubation in the presence of antiserum to hCG or to hLH. In our study, however, a doseresponse curve was obtained with ovine FSH which was parallel to that for ovine LH, but was displaced toward higher concentrations (2000x). Studies with rabbit anti-ovine LH serum were conducted to determine if the FSH response was intrinsic or due to LH contamination. Results of these studies are presented in Fig. 1. It is seen that the addition of LH antiserum to the LH (200-500 /.q of LWml of AS) completely inhibited the production of testosterone (Fig. 1A). Addition of normal rabbit serum did not affect the LH response. When ovine FSH was assayed with LH antiserum (1 and 5 mg of FSWml of AS), there was a partial reduction (33%) in the response when compared to FSH alone (Fig. 1B). Thus, while there appears to be a trace of LH in the FSH preparation, the response to the antiserum-treated FSH was dose dependent and significantly higher than the controls in each case, and it prob-
r
- 60 ’ t -4of
:: t -20 @ @
FIG. I. The effect of LH antiserum dig cells by LH (A) and FSH (R).
on the stimulation
of testosterone
production
in isolated rat Ley-
490
FARMER,
SUYAMA,
ably represents a small but intrinsic activity of the FSH.’ Mammalian
LHs
The stimulation of testosterone production by isolated Leydig cells with highly purified LHs from eight mammalian species, as well as with hCG and PMSG, is shown in Fig. 2. The data in the left panel are from a single experiment, and those in the right panel are from four experiments which showed an identical response for ovine LH [G3-222B (Papkoff et al., 1965)l. This ovine LH was run in all experiments as a standard; Fig. 2 (right panel) also includes the response of another standard, NIH-LH-S18. All of the LHs gave parallel log dose-related responses, with the same maxima when high enough doses were assayed. Averaged potency estimates for these hormones are listed in Table 1. There was a very wide range of potencies (250fold) for the various LHs, all of which are of comparable purity. Species variation can 2 Bovine TSH was also found to stimulate testosterone production in a manner comparable to ovine LH at a high concentration (500x ovine LH), but the specificity of this response was not checked with antiserum.
AND
PAPKOFF
also be seen with the same preparations assayed in both the ovarian ascorbic acid depletion (OAAD) assay (fivefold range) and the Xenopus ovulation assay (lOO-fold range) [Table 1, Licht and Papkoff (1976)l. There was no consistent relationship in potency rank among the species, but the results of the two mammalian assays were the most similar. Nonmammalian
Gonaa’otropins
LHs from nonmammalian species also stimulated testosterone production in rat Leydig cells in a manner identical to that of ovine LH, but at approximately a lOOO-fold greater dose level. These results are shown in Fig. 3, with LHs from the turkey, alligator, snapping turtle, sea turtle, and bullfrog. The potencies of these various LHs were quite similar; there was only a six-fold difference, compared with the 250fold potency range observed with LHs from mammalian species (Table 2). FSHs from these same nonmammalian species were assayed at one or two dose levels. They were all active, at higher dose levels than the respective LHs, but were much closer in potency to their LHs than were mammalian FSHs. These data are summarized in
iQo=” 60. 2 % \ 60t H 40!! E B m-
HORMONE
FIG. 2. Stimulation d testosterone production from eight mammalian species.
CONCENTRATION
(ng)
in isolated rat Leydig cells by hCG, PMSG, and LHs
EFFECT
OF GONADOTROPINS TABLE
BIOLOGICAL
ACTIVITIES AND
Species hCG Human LH Horse LH PMSG Sheep LH Ox LH Pig LH Rat LH Rabbit LH Dog LH
AND
SEVERAL
491
CELLS
I
CARBOHYDRATE
CONTENT
MAMMALIAN
Total carbohydrate content a 34 28 24 45 16 13 14 15 ND ND
ON LEYDIG
OF
hCG, PMSG,
LHs Biological
Leydig’
25.3 (17.3-35.2) 9.7 (6.8-14.0) 6.3 (2.7-l 1.O) 2.6 (I .9-3.8) 1.6 (1.1-2.6) 0.9 (0.5-I .5) 0.4 (0.2-0.6) 0.4 (0.2-0.7) 0.2 (0.1-0.5) 0. I (0.04-0.2)
potencies
OAADd NDe 4.1 3.0 1.5 2.8 1.0 1.2 1.0 0.8 ND
*
Xenopus
ND 0.05 0.6 ND 2.8 1.3 0.1 <0.25 0.025 ND
a Grams/l00 g of protein. * All potency estimates are x NIH-LH-Sl. ’ Averaged potency values; 95% confidence limits are in parentheses. d Taken from Licht and Papkoff (1976). e ND, not determined.
Table 3, where the potencies of the various species of FSH are given in terms of the LH from the same species. Thus, while ovine FSH is active only at levels 2300x greater than ovine LH, snapping turtle FSH is equally active at levels only 17x greater than snapping turtle LH. DISCUSSION
The data presented here show the response of isolated Leydig cells to a wide variety of mammalian and nonmammalian LHs. All of these LHs were prepared by similar methodology and are of comparable purity, facilitating comparisons. The range of species studied includes at least one representative species from each tetrapod class and representatives of six different mammalian orders. Similar results with some of these hormones have been reported previously by Ramachandran and Sairam (1975) and Dufau et al. (1976). The fact that all of these species of LH, as well as hCG and PMSG, were able to stimulate testosterone production in rat Leydig cells attests to the basic similarity of these LHs and the wide applicability of the assay.
The insensitivity of one species to the hormones derived from a different species is termed species specificity. It is known that the structures of both the hormone and the target cell receptor play a role in determining species specificity. In our study, the use of an in vitro assay eliminated the problem of differences in the metabolic clearance of the hormones, and thus the variation in potency observed among the LH species (250-fold) must relate to structural differences which affect their interaction with the target tissue. The primary structures of hCG and five of the LHs employed are known and the differences in amino acid sequences among these species could account for the potency differences observed. In addition, in our studies a high content of carbohydrate appeared to correlate well with high activity (Table 1). The relationship of carbohydrate and hCG potency has been directly studied by Moyle et al. (1975). They observed that the removal of sugar residues from hCG caused a reduction in potency in the rat Leydig cell assay while parallelism and the response maxima were not affected. The response of PMSG
d
492
FARMER,
P
SUYAMA,
AND
PAPKOFF
20.
0
I WI
lb,
I
HORMONE
CONCENTRATION
FIG. 3. Stimulation of testosterone production in isolated rat Leydig cells by sheep LH and LHs from five nonmammalian species. Note different concentration ranges employed for the mammalian (nanogram) and nonmammalian (microgram) LHs.
TABLE
2
BIOLOGICAL ACTIVITIES OF NONMAMMALIAN LEYDIG CELL ASSAY AND THE Xenopus
LHs MEASURED OVULATION
Bioloeical
Turkey Alligator Snapping turtle Sea turtle Bullfrog
2.6 I.1 I. I 0.5 0.8
a Taken from Farmer et al., TABLE
1975; Licht er a/., 3
BIOLOGICAL POTENCIES OF SEVERAL SPECIESOF FSH MEASURED AGAINST THE SAME SPECIES OF LH IN THE RAT LEYDIG CELL ASSAY
Sheep ox Turkey Alligator Sea turtle Snapping turtle Bullfrog
Potency (x LH of same species) 0.005 0.005 0.36 0.03 0.07 0.06 0.26
Xenopus
x NIH-LH-S
(I .8-4.2) (0.8-1.3) (0.8-I .4) (0.4-0.6) (0.5-I .2)
1976a; Papkoff et al.,
RAT
uotencies
Leydig lO-3 x NIH-LH-Sl (95% limits)
LH obtained from
FSH obtained from
IN THE ASSAY
a
1
0.3 3.3 1.8 I.0 0.3 1976a,b.
is notable because this aonadotroninI has the highest carbohydrate content but was ranked only fourth in Leydig cell potency (Table 1). This is in part due to the fact that the potency calculations employed nanogram per milliliter hormone concentrations rather than molarity, since the precise molecular weights of most of the hormones employed are not known. In addition, PMSG is known to possess both LH and FSH activities and may therefore have an amino acid sequence somewhat different from the sequences of the other LHs
EFFECT
OF GONADOTROPINS
tested, which may also account for its relative potency. Data obtained with the same LH preparations in two other bioassays, the OAAD and Xenopus ovulation, were also shown in Table 1. The potency differences observed for each hormone in the Leydig and OAAD assays are interesting in that comparisons are made for responses which were obtained in vitro versus in vivo and in male versus female and for a physiological parameter (testosterone production) versus a response of unknown significance (ascorbic acid depletion). Any of these factors could account for the LH potency differences observed between the two assays. The potency ranking, however, is similar in these two rat assays and quite different from that seen in the frog assay. This comparison supports the idea that the receptor as well as the hormone play a role in determining species differences. In the past it has proven difficult to assay nonmammalian gonadotropins in in vivo mammalian assays. With the use of sensitive in vitro assays, however, both we and Licht et al. (1976b) have been able to demonstrate an effect of nonmammalian gonadotropins on mammalian tissue, although lOOO-fold greater dose levels were necessary. The nonmammalian LHs are of purity comparable to that of the mammalian hormones , as judged by chemical criteria (Farmer et al., 1975; Papkoff et al., 1976a; Licht et al., 1976a) and by their potencies in the Xenopus ovulation assay (Table 2). Therefore, there is indeed great specificity of rat Leydig cells for mammalian hormones. Nevertheless, because of its sensitivity, this assay can be used to advantage in studies on nonmammalian hormones. LH specificity in the Leydig cell assay was retained with the nonmammalian hormones. However, the potency differences between the nonmammalian LHs and FSHs were much closer than those found with the mammalian hormones (Table 3). It is of interest that mammalian FSH and TSH give
ON LEYDIG
CELLS
493
dose-responses at the same concentrations at which nonmammalian gonadotropins are active. These data could suggest that, at these high hormone concentrations, the Leydig cell begins to respond to structural features which the glycoprotein hormones all have in common. In conclusion, our results demonstrate the usefulness of the rat Leydig cell assay for structure-function studies. In such studies, the variety of species of LH serve as natural analogs, since preparation of synthetic analogs of these large glycoproteins is as yet impossible. Our data confirmed the high specificity of the Leydig cell assay for mammalian LHs, which are active at nanogram levels, but also showed that, at microgram hormone levels, the Leydig cell recognizes more general glycoprotein features and responds to mammalian FSH and TSH and to nonmammalian LHs and FSHs. ACKNOWLEDGMENTS We thank Professor Choh Hao Li for his support of these studies and for critically reading the manuscript. This work was supported in part by a grant from the National Science Foundation, BMS 7516138, a Rockefeller Foundation Center Grant, and NIH Grants AM-6097 and HD-05722.
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Farmer, S. W., Papkoff, H., and Licht, P. (1975). Purification of turkey gonadotropins. Biol. Reprod.
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Finney, D. J. (1964). “Statistical Methods in Biological Assays,” pp. 99-163. Griffen, London. Licht, P., and Papkoff, H. (1976). Species specificity in the response of an in vitro amphibian (Xenopus laevis) ovulation assay to mammalian luteinizing hormones. Gen. Comp. Endocrinol. 29,552-555. Licht, P., Farmer, S. W.. and Papkoff, H. (1976a). Further studies on the chemical nature of reptilian
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gonadotropins: FSH and LH in the American alligator and green sea turtle. Biol. Reprod. 14, 222-232. Licht, P., Mullet-, C. H., and Tsui, H. W. (1976b). Effects of mammalian and nonmammalian gonadotropins on andtogen production by minced rabbit testis. Biol. Reprod. 14, 194-201. Moyle, W. R., and Ramachandran, J. (1973). Effect of LH on steroidogenesis and cyclic AMP accumulation in rat Leydig cell preparations and mouse tumor Leydig cells. Endocrinology 93, 127-l 34. Moyle, W. R., Bahl, 0. P., and MBrz, L. (1975). Role of the carbohydrate of human chorionic gonadotropin in the mechanism of hormone action. J. Biol. Chem. 250, 9163-9169. Papkoff, H. (1973). Studies on the structure of ovine follicle stimulating hormone. In “Proceedings, 4th International Congress of Endocrinology, Washington.” Excerpta Medica Znt. Congr. Ser. 273, 596-600. Papkoff, H., and Can, J. (1970). Bovine interstitial cell-stimulating hormone: Purification and properties. Arch. Biochem. Biophys. 136, 522-528.
AND PAPKOFF Papkoff, H., Gospodarowicz, D., Candiotti, A., and Li, C. H. (196.5). Preparation of ovine interstitial cell-stimulating hormone in high yield. Arch. Biochem. Biophys. 111, 431-438. Papkoff, H., Farmer, S. W., and Licht, P. (1976a). Isolation and characterization of folliclestimulating hormone and luteinizing hormone and its subunits from snapping turtle (Chelydra serpentina) pituitaries. Endocrinology 98, 767-777. Papkoff, H., Farmer, S. W., and Licht, P. (1976b). Isolation and characterization of luteinizing hormone from amphibian (Rana catesbeiana) pituitaries. Life Sci. 18, 245-250. Ramachandran, J., and Sairam, M. R. (1975). The effects of interstitial cell-stimulating hormone, its subunits, and recombinations on isolated rat Leydig cells. Arch. Biochem. Biophys. 167, 294300. Schams, D., and Papkoff, H. (1972). Chemical and immunochemical studies on pregnant mare serum gonadotropin. Biochim. Biophys. Acta 263, 139148.