In vitro stimulation of 5 α-dihydrotestosterone and testosterone secretion from bullfrog testis by nonmammalian and mammalian gonadotropins

GENERALAND

COMPARATIVE

ENDOCRINOLOGY

33, 10%121 (1977)

In Vitro Stimulation of 5 a-Dihydrotestosterone and Testosterone Secretion from Bullfrog Testis by Nonmammalian and Mammalian Gonadotropins CHARLES H. MULLER~ Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720 Accepted March 18, 1977 In vitro androgen secretion by bullfrog (Rana catesbeiana) testis was studied using mammalian and nonmammalian gonadotropins, including those from the bullfrog. LH from bullfrog, salamander, turtle, alligator, and sheep increased secretion of Sodihydrotestosterone (DHT) and testosterone (T) into incubation medium; Rana and ovine FSH preparations were inactive except at relatively high concentrations. Rana FSH, ovine PRL, and bovine GH did not influence LH-induced steroid secretion. Rana LH was about 3- 10 times more potent than other nonmammalian LH preparations, and 30-50 times more potent than NIH-ovine LH. Dose-response characteristics were dependent upon incubation time and temperature. More DHT than T was present in incubation medium at ah times and temperatures studied, and during all seasons. Contents of DHT and T in incubation medium were highly correlated, and the ratio DHT/T increased with time and with dose of LH. These data support in vivo observations and indicate that DHT is a major testicular androgen in the bullfrog, stimulated specifically by LH. Control of testicular androgen secretion by LH may represent the ancestral tetrapod condition.

Stimulation of testicular androgen secretion in vitro by gonadotropins has been demonstrated in all classes of tetrapod vertebrates. Recently, in vitro steroid secretion by gonadal tissues was studied in terms of gonadotropic specificity in attempts to elucidate evolutionary patterns of gonadotropin structure and function (Licht et al., 1977). Androgen production and secretion by the mammalian testis, in vivo and in vitro, are LH-specific responses, although other hormones (notably FSH and prolactin) may have modulating roles (cf. Johnson and Ewing, 1971; Hafiez et al., 1972; Catt and Dufau, 1973; Ode11 et al., 1973; Cooke et al., 1974; Bartke and Dalterio, 1976; Chen 1 Send reprint requests to Cancer Research Laboratory, University of California, Berkeley, California 94720. (Present address of author: Department of Zoology, University of Texas, Austin, Texas 78712.)

et al., 1976). Mammalian FSH, when tested alone, is without effect, or its effect can be largely ascribed to LH contamination. These results demonstrate that a variety of testis preparations from the few species studied respond to mammalian LH with a high degree of specificity. Testosterone secretion from rat Leydig cells is stimulated by bullfrog LH (Papkoff et al., 1976a), and testosterone secretion from minced rabbit testis is stimulated by bullfrog LH and other nonmammalian gonadotropins (Licht et al., 1976b). In both studies, bullfrog LH has only about 0.1% the potency of mammalian LH. In the last study, a loss of gonadotropic specificity was observed in some cases when nonmammalian gonadotropins were tested, i.e., FSH was almost as potent as LH for some species. In contrast to the mammalian situation, in vitro steroid secretion by the reptilian 109

Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISSN 00164480

110

CHARLES

testis is, in general, stimulated by both LH and FSH. The LH/FSH potency ratio varies with both the source of testis and the source of gonadotropins (see Tsui, 1976a; Tsui and Licht, 1977; Licht et ul., 1977). The circumtesticular Leydig cells of the lizard Cnemidophorus tigris respond equally to ovine FSH and LH (Tsui, 1976b). Snake and turtle testes studied respond almost equally to LH and FSH, especially when homologous hormones are tested (see Licht et al., 1977). In terms of goMdotropic specificity for in vitro androgen secretion, reptiles differ considerably from mammals, the former exhibiting a relative lack of specificity. These observations raise questions concerning the nature of gonadotropic specificity in the ancestral tetrapod stock. Therefore, it was considered of interest to examine the gonadotropic specificity of in vitro testicular androgen secretion in the oldest tetrapod class, the Amphibia. Results of in vivo studies in the bullfrog suggest that testicular androgen secretion is an LH-specific response (&fuller, 19762, 1977). This conclusion supports the indirect in vivo evidence that mammalian LH, but not FSH, stimulates androgen production in Rana pipiens (Burgos and Ladman, 1957) and Rana temporuria (Lofts, 1961). However, A5-3phydroxysteroid dehydrogenase (3PHSD) activity (measured in vitro) is greatly increased after injection of Xenopus luevis with either mammalian LH or FSH; indeed, FSH is re~~edly more potent than LH (Wiebe, 1970). Since the three last reports dealt with heterologous hormones and indirect evidence of androgen production, further investigation is required. The present study is concerned with androgen secretion by the bullfrog (Rana catesbeiuna) testis in vitro during incubation with homologous and heterologous LH and FSH preparations. Preliminary reports of portions of this work have appeared earlier (Muller, 1975, 1976a, b).

H. MULLER

MATERIALS Preparation

AND METHODS

and ~ncu~ution

of Testis

Adult male R. catesheiana from central California were obtained from commerical sources at various times over a 2-year period (1974-1976) and kept in the laboratory 3 days to 2 weeks before use. Frogs were decapitated and testes were quickly excised and placed into ice-cold incubation medium (see below). Fat bodies and mesorchia, including vasa efferentia, were removed and the testes were decapsulated as completely as possible. In preliminary experiments whole-, half-, and quarter-testes, l-mm-thick slices, and I x I- to 2-mm pieces were tested: positive resuits (LH-stimulated steroid secretion) were obtained in all cases. Consequently, in order to test several different hormones over wide dose ranges, most experiments employed testis pieces prepared by transversely slicing the testis with a razor blade; the l-mm slices were then cut with razor blade or scissors into 1 X I- to 2-mm pieces (representing 100-500 m of protein). Slicing was done in incubation medium over ice, and medium was changed once or twice during the procedure, which was completed within 1 hr after the animal was killed. Each testis of a large frog yielded 30 to 50 pieces. In most experiments, testis pieces from only one frog were used, but for incubations involving over 100 tubes, testis pieces were either pooled from several frogs or kept separately, with replicate tubes representing different frogs. Testis pieces were transferred to a small Erlenmeyer flask and washed three times with fresh cold medium. Flasks were placed in a 28-30” shaking water bath. gassed with 95% 02-5% CO*, and preincubated for 30-60 min. After preincubation, flasks were cooled on ice and washed three times with fresh cold medium. Pieces were kept on ice until incubation was started. Dilutions of hormones (usually in 0.25 or 0.5 ml of incubation medium) were added to each 12 x 75-mm glass culture tube, and one testis piece was blotted and then placed into the medium for each tube. Tubes were incubated in a shaking (2 cps) water bath under 95% 02-5% CO,, usually for 2 hr at 2830”. Responses at different times and tem~ratu~s were also studied, as described in Results. After incubation, tubes were chilled in ice water and centrifuged at 4” (1700 g for 10 mitt), and a portion of the medium was frozen at -20” or assayed immediately. Testis pieces were dried in a 56” oven, defatted in ether:chloroform:methanol (1 : I : I), and digested in IN NaOH for protein determination (Lowry et al., 1951). In preliminary experiments, amphibian Ringer solution (a poorly buffered saline isotonic with amphibian plasma), Krebs-Ringer-bicarbonate buffer (KRB; an incubation medium developed for mammalian tissue),

BULLFROG and medium 199 (a defined mammalian culture medium containing nutrients and cofactors) were used as incubation media. Stimulation of androgen secretion by LH was obtained with all these media. Results showed that tissues responded equally (in terms of baseline, LH sensitivity, and maximum androgen production) when incubated in amphibian Ringer or in KRB. The KRB was chosen for further work because of its buffering capacity and extensive use in similar studies of mammaljan and nonmammalian (including amphibian) steroidogenesis. The KRB (pH 7.4) had the following constituents (m&f): NaCl, 118.0; KCI, 4.8; CaCI,, 2.5; MgSO,. 1.17; KH,PO,, 1.2: NaHCQ, 25.0. In some cases gelatin (IO pg/ ml) was added to decrease possible adsorption of hormones to incubation tubes; glucose and organic substrates were not included in the medium.

TESTIS IN ViTRO

Ill

FSH bioassay were 2.4, 5.5. and 5.7 x NIH-FSH-Sl. respectively. In Xenoprrs ovulation assay. C6IB had 0.3% bullfrog LH activity (P. Licht, unpublished); C61 B and C60B had l-3% immunoactivity in bullfrog LH RIA (Daniels et a/., 1977). Tiger salamander (A~~~~~u~a ~~g~j~r{~ 1. snapping turtle (Chelydra serpentina), and sea turtle (Chehia mydas) and Alligafor mississipiensis gonadotropins have been described by Licht et al. (1975). by Licht and Papkoff (l974b) and Papkoff et al. (l976bl. and by Licht et ul. (1976a), respectively. Ovine LH (NIH-LH-Sl7 and -SlS), ovine FSH (NIH-FSH-SIO), ovine prolactin (NIH-P-S9 and -SlO). and bovine growth hormone (NIH-GH-B8 and -Bl6) were obtained from the Hormone Distribution Program of the National Institutes of Health.

Statistics Radioimmunoassay

of Androgens

The radioimmunoassay (RIA) procedure for 5~ dihydrotestosterone (DHT) and testosterone (T) has been described in detail elsewhere (Licht and Tsui, 1975; Licht er al., 1976a; Muller, 1976~). The incubation medium was extracted and chromato~aphed prior to RIA as previously described, or was assayed directly (diluted at least 1 : IO in RIA buffer). For direct RIA, DHT was used as tracer and standard; results are expressed as “total” DHT f T, although the T component is overestimated by 45.5% and unknown cross-reacting materials may alter quantitative results. However, correlation between chromatographed DHT and “total” DHT + T in the same sample was highly significant (P < 0.01; r = 0.99) and chromatographed DHT plus T accounted for approximately 88% of “total” DHT + T by direct RIA. Furthermore, direct assay of incubation medium yielded inhibition curves parallel to the standard curve. The identity of DHT in incubation medium containing bullfrog testis has been verified by thinlayer and gas-liquid chromatography (Muller, 1976~).

Hormones Bullfrog gonadotropins were purified and assayed as described by Licht and Papkoff (1974a); the bullfrog (Rann) LH (C53C and ClS3C) was further characterized by Papkoff et al. (1976a). This LH is 0.33 x NIH-LH-S 1 potency in Xenopus ovulation assay, contains 2% of Runa FSH activity in Anolis bioassay (Licht er al., 1977). but only 0.4% Rana FSH immunoactivity by homologous FSH RIA (Daniels et al., 1977). Three Rana FSH preparations were tested, one of intermediate purity (C6lB) and two more highly purified preparations (C60B and CVSA). Potencies of C6lB, C95A. and C6OB in Aplofis

Potency estimates were obtained by graphical analysis and by parallel-line assay statistics (Finney, 1964). Analysis of variance, multiple range tests, regression analysis, and factorial analysis were conducted using the EXBIOL program (Sakiz) on a CDC 6400 computer at the University of California, Berkeley.

RESULTS Gonado~ropic Secretion

~pec~~c~~ of Androgen

Rana gonadotropins were tested repeatedly, during different seasons, to establish the gonadotropic (LWFSH) specificity of in vitro stimulation of androgen secretion. Rana LH was tested in 15 experiments over a Z-year period. In six experiments, Rana LH stimulated increased androgen secretion, but Rana FSN was not tested. Rana LH and FSH were compared in nine of the experiments, conducted in January, February, March, April, June, August, and October. In eight of the nine experiments, Rana LH stimulated significant increases in DHT and T, while Rana FSH had no effect; results of one of these experiments are illustrated in Fig. 1. In the remaining experiment (done in February) basal secretion levels were unusually high, and none of the gonadotropins tested stimulated increased secretion. In another experiment, wide dose ranges (0.05-410

112

CHARLES

FIG. 1. Comparison slices (representing 3-4 for 3 hr at 30” in KRB Each point and vertical

H.

MULLER

of effects of Rana LH and FSH on DHT secretion by bullfrog testis in vitro. Testis mg of protein) were from four bullfrogs captured in late August. Slices were incubated alone (control) or in 1 ml of KRB containing Ram LH (C53C) or Rana FSH (C6OB). line represents the mean and SE for four replicate tubes.

&ml) of Rana LH, FSH, and crude pituitary extract were compared (Fig. 2). In this case, Runu FSH at high doses (> 100 pg/ml) induced minimal stimulation of androgen secretion; this activity ofRunu FSH was equivalent to O.l-0.3% that of Runu

LH. The potency ofRana LH was lo-25 x alkaline extract of Runu pituitary. In two experiments, addition of Runu FSH to incubation medium had no effect on the response to Runu LH. For example, in the experiment illustrated in Fig. 1, SO0 ng

35'0h

30-

E" 2% :

+

!z n m =

20'5IO5O.OS 0.1 0.2 0.4 0.6

1.6 12

6.4

12.8 S6 92 IO24 2046 40%

FIG. 2. Stimulation of in vitro androgen secretion by Rana LH, FSH, and pituitary extract. Pieces of testis (representing 100-500 pg of protein) were from one frog captured in October. Testis pieces were incubated for 2 hr at 28-30” in 0.25 ml of KRB done or in medium containing Rana LH (C53C), Ra?Ia FSH (C61B), or&no pituitary extract (ClE). The bar and vertical line represent the mean and SE of six control tubes. The points are means of two tubes for each dose of hormone.

BULLFROG

of Rana FSH/ml were added to each dose of Rana LH; the dose-response curve under these conditions was identical to the one illustrated for LH alone. Heterologous gonadotropins (ovine and nonmammalian), and mammalian PRL and GH were also tested. In three tests, incubation of bullfrog testis pieces with ovine PRL or with bovine GH (NIH-GH-B8), alone (O.Ol- 1 &ml) or in combination with Rana LH, had no effect on androgen secretion or on the LH-induced response. A different GH preparation (NIH-GH-B 16) exhibited slight LH-like activity. Ovine LH stimulated androgen secretion (with a doseresponse curve parallel to that of Rana LH), whereas ovine FSH produced a slight, nonparallel response at high doses (>50 E.Lg/ml). In three tests, the potency of Rana LH was 30-50 x NIH-LH (Fig. 3). Alligator, Ambystoma, and Chelydra LH were active, but their potencies in three tests were 0.1-0.4 x Rana LH. Chelonia LH, Chelonia FSH, and Alligator FSH were not active at doses tested, indicating that their potencies were <0.08 x Rana LH. Arnbystoma and Chelydra FSH preparations were not tested.

.5 0) is h E”

113

TESTIS IN VITRO

Characteristics

of LH-Induced

Response

Incubation of tissue alone (without hormones) established basal secretion levels, which generally ranged from 1-2 ng of DHT + T/mg of protein for 2 hr at 28-30”; however, testis tissue from some frogs (in February and October) secreted 5-8 or more ng/mg for 2 hr. Sensitivity, precision, maximum response, and the nature of the dose-response curve varied considerably among experiments and, in some cases, among frogs in a single experiment. This variation is reflected by indexes of precision which generally ranged from 0.16 to 0.30. Sensitivity (lowest LH dose significantly stimulating androgen secretion over control levels) ranged from ~100 rig/ml to about 1 &ml, but was generally between 100 and 200 rig/ml. Maximum responses were usually >25 ng of DHT + T/mg of protein, with individual samples reaching 50 or sometimes >lOO ng/mg. Thus, 25 to lOO-fold increases over basal levels were observed. In a few cases, however, both basal and maximal levels were considerably lower than usual (e.g., Fig. 3). The maximum response in some cases was in the form of a plateau over a lo-fold LH

1.0 -

P /’ ‘,

OXI-

/# ’

/ b

. c g 0

os-

P 0.25-

‘\

*

1 ‘d

CONTROL 0.1

I 0.2

0.4

0.0

I.5

pg

3.2

6.4

IO

16

32

1 64

LH/ml

FIG. 3. Stimulation of in vitro androgen secretion by Ram LH and ovine LH. Testis pieces were from one frog collected in September; incubation conditions and symbols are the same as those in Fig. 2.

114

CHARLES

H. MULLER

dose range, or in others reached a peak. In affected basal secretion levels and sensitiveither case, inhibition of androgen secre- ity to LH. In one experiment (in February), tion was observed when sufficiently high testis pieces from one frog were randomly doses of hormone were tested (cf. Figs. 2 separated into three groups, and each group and 3). Analysis of chromatographed sam- (consisting of control and LH tubes) was ples revealed that secretion of both DHT incubated at a different temperature. Reand T was inhibited at high doses of Rana sults are presented in Fig. 5A. All three LH or pituitary extract. doses of Rana LH were stimulatory at 26”, Incubation time and temperature influ- but had no effect at either 16 or 36“. Addienced certain aspects of the response. All tionally, basal levels were highest at 26”. In results reported above were from 2-hr in- a second experiment (in April), incubations cubations at 28-30”. Incubations carried were carried out at 23 or 32” for 2 and 4 hr. out for 30 min to 3 hr demonstrated that Results, illustrated in Fig. 5B, revealed both T and DHT contents of the incubation two- to three-fold shifts to the right in the medium increased with time. Increases in response curve with either a 9” decrease in androgen content were observed in con- temperature or a halving of incubation trols, and were greatly influenced by LH, time. Sensitivity, basal levels, and response as illustrated in Fig. 4. Additionally, sen- at a given dose were greatest at 32”, 4 hr sitivity was increased (lower doses of and most reduced at 23”, 2 hr. LH produced significant stimulation) with Relationship of DHT to T increased time (see Fig. 4). As has been demonstrated (Muller, Incubation at different temperatures also 1976c), T in the incubation medium can be converted to DHT. More DHT than T was present in incubation medium under all Rana cafesbeionatestis conditions (including time, temperature, presence of different hormones, season), and DHT and T were highly correlated (typically, Y = 0.9). Both DHT and T accumulated with time, as shown in Fig. 6, although DHT accumulated at a more rapid rate than T (difference between slopes in Fig. 6, P < 0.05). Dose-response curves for T were poorer than those for DHT in terms of LH sensitivity, slope, and maximum response. Furthermore, as seen in Table 1, the ratio DHT/T increased significantly not only with time (P < O.Ol), but also with increasing LH dose (P < 0.01); the interaction between LH and time was significant (P < 0.05). Incubation with FSH had no effect (P > 0.05) on DHT/T (Table 2). FIG. 4. Effects of incubation time and Rana LH on in vitro DHT secretion. Data are from the experiment described in Fig. I. Samples were obtained after 30, 90, or 180 min of incubation. Testis slices were incubated with KRB alone (0) or with 20, 100, or 500 ng of LWml (LHZO-LH5,&. Each point and line represents the mean and SE of four replicate tubes.

DISCUSSION Effects of Incubation Temperature

Temperature is an important factor regulating reproductive cycles of temperate

BULLFROG

TESTIS

IN

115

VlTRO

5

d Ii pug Rum LH/ml

FIG. 5. Effects of incubation tem~rature and time on in vitro androgen secretion. Points illustrate the mean androgen content of three rephcate tubes; bars and vertical lines are mean and SE for androgen content of three (A) or four (B) control tubes (KRB without LH). Data in (A) are from a February experiment; testis pieces from one frog were incubated for 4 hr at 16,26, or 36”. Data in (B) are from an April experiment; pooled testis pieces from 1I frogs were incubated for 2 or 4 hr at 23 or 32”. Symbols over bars representing control v&es are: a = 2 hr, 23”; b = 4 hr, 23”; c = 2 hr, 32”; d = 4 hr, 32”.

TABLE EFFECTS RATIO

I

OF Rana LH AND TIME ON DHWT DURING IMXJBATION OF BULLFROG TESTIS

Incubation

DHT /

Ram

time (min)

LH 30

Wml) 0 20 loo so0

rnn

2.4 3.3 2.0 2.8

k I 2 k

90 0.3” 0.5 0.3 03

4.3 3.2 3.8 5.7

It 0.4 t- 0.5 L 0.2 -c 0.4

I80 5.3 2 5.4 f 6.7 + 10.7 t

0.6 I.2 0.6 I3

* Mean i SE of four replicate tubes. Factorial analysis ofRana LH dose-response, P < 0.01; time effect, P < 0.01; interaction of LH and time, P i 0.05.

0

kz

142 Hours

3

FIG. 6. Effect of iucub~on time on accumulation of T and DHT in incubation medium. See Figs. I and 4 for a description of the experiment. Each point and vertical fine represents the mean and SE of four replicate tubes.

zone ectotherms. Seasonal testicular changes in anuran amphibians have been studied extensively (see Lofts, 1974), with particular regard to temperature. In Runa esculenta and R. t~~p~r~r~a, high environmental temperatures (up to 26”) stimuIate s~rmatogenesis and induce regression of thumb pads, seminal vesicles, and

116

CHARLES

H.

TABLE 2 EFFECTS OF Rana FSH AND TIME ON THE RATIO DHT/T DURING INCUBATION OF BULLFROG TESTIS Incubation

time

(min)

Rana FSH 30

(w/ml) 0 mo 2500 12500

2.4 4.2 3.4 3.6

” 2 k f

90 0.3” 1.9 0.5 0.6

4.3 4.7 4.0 3.6

2 2 + 4

180 0.4 0.8 0.2 0.2

5.3 6.4 5.9 6.0

’ Mean + SE of four replicate tubes. Factorial ysis of Rana FSH effect, P > 0.05; time effect, 0.01; interaction of FSH and time, P > 0.05.

2 f k ”

0.6 1.2 1.2 0.8 analP <

Leydig cells, as judged by histological and histochemical criteria (van Oordt, 1960; van Oordt and Lofts, 1963; de Kort and van Oordt, 1965; de Kort, 1967; van Kemenade, 1969). At both high (24”) and low (4”) temperatures, injections of ovine LH into R. esculenta in winter induced an increase in Leydig-cell 3fl-HSD and in Leydig-cell nuclear diameter, but the effect at 4” was greater than that at 24” (de Kort, 1967, 1971). Treatment with LH in winter at 4” stimulated thumb pad development, but such treatment at 24” had no effect on thumb pads. Partly on the basis of the above results, it has been postulated that the sensitivity of the two testicular functions to gonadotropin varies with environmental temperature: Spermatogenic activity is stimulated at high temperatures, and androgen secretion at low temperatures (van Oordt and de Kort, 1969; de Kort, 1971). The results of the present report do not support this proposition for the bullfrog. The incubation temperature of 28-30” was chosen because bullfrogs behaviorally thermoregulate at about this temperature in nature during the breeding season (Lillywhite, 1970). Stimulation of in vitro androgen secretion by LH at 28-30” was observed during all seasons. Further, in two experiments (in February and April), incubation at lower temperatures (16 or 23”)

MULLER

resulted in significant decreases in androgen secretion, and relative loss of LH sensitivity. Discrepancies between results in the present report and those of de Kort (1971) may lie in differences between species or experimental approaches. In particular, de Kort (1971) and others used thumb pad stimulation as an index of in vivo androgen secretion, and this response may be subject to temperature-dependent changes in sensitivity to androgens, and to temperature-dependent changes in the metabolic clearance rate of secreted androgens. In the present report, a decrease in androgen secretion and loss of LH sensitivity were also observed at a higher temperature (36”). This result corresponds to the lethality of this temperature for bullfrogs (Lillywhite, 1971). This effect of high temperature on bullfrog testicular steroidogenesis is reminiscent of the situation in mammals. For example, Levier and Spaziani (1968) observed that scrotal temperature (32”) was optimal for in vitro steroidogenesis by rat testis; at 28 and 36” in vitro steroidogenesis was significantly impaired. Secretion of DHT by Bullfrog Testis In vitro incubation of testes from a wide variety of vertebrates has provided important information concerning phylogenetic and seasonal differences in steroidogenic capabilities. Studies of conversion of exogenous labeled precursors, or isolation and identification of endogenous steroids, have indicated testosterone to be a major testicular androgen in fish, amphibians, reptiles, birds, and mammals. Additionally, the presence of androstenedione, progesterone, and estrogens in testes of certain representatives of all vertebrate classes (see reviews by Eik-Nes, 1971; Chieffi, 1972; Ozon, 1972) suggests a certain degree of evolutionary conservatism in testicular steroidogenesis, but qualitative and quantitative differences from the generalized mammalian scheme should be expected. For example, 1 1-ketotestosterone is a

BULLFROG

TESTIS

major testicular steroid in some fish (see Idler et al., 1971). However, testicular steroids of vertebrates have not been studied so extensively as adrenocortical steroids, which exhibit both differences and similarities between and within vertebrate classes (see Sandor, 1969). The secretion of relatively large amounts of DHT by the testes of R. catesbeiuna represents one such divergence from the mammalian pattern, although small amounts of DHT and other ring A reduced steroids are produced by adult mammalian testes, and are present in low amounts in mammalian plasma (see Eik-Nes, 1975). Using identical incubation, chromatography, and radioimmunoassay techniques, testes from rabbit and various birds and reptiles were found to produce little or no DHT in vitro (Licht et al., 1976b; Tsui, 1976a; Tsui and Licht, 1977). The identification of testosterone in testis of the bullfrog was first reported by Dale and Dorfman (1967), but these authors did not isolate other androgens. However, DHT is a major metabolite of T during in vitro incubation of testes from the anuran amphibians R. temporaria (Ozon et al., 1964), Discoglossus pictus (Ozon and Stocker, 1974), Nectophrynoides occidentalis (Gavaud, 197S), and R. catesbeiana (Mullet-, 1975. 1976~). In addition, more DHT than T is present in plasma of adult male R. catesbeiana at certain periods of the reproductive cycle (Muller, 1976b,c), and after injection of homologous LH (Mullet-, 1977). 5aReductase activity has also been found in anuran amphibian liver (Lisboa and Breuer, 1966), seminal vesicles, thumb pads, and skin (Ozon and Fouchet, 1972). Therefore, at least for some amphibian species, it is proposed that considerable formation of DHT occurs within the testis, that both T and DHT are secreted by the testis, that more DHT than T is present in peripheral plasma during some phase of the reproductive cycle, and that further reduction of T to DHT occurs in certain androgen-

IN

VITRO

117

responsive tissues. The cellular site of 5~ reductase in the amphibian testis has not yet been resolved; attempts to isolate interstitial and tubular tissues of bullfrog testis have been unsuccessful to date (C. Muller, unpublished). Although T is a potent androgen in anurans, few studies have compared its effects with those of DHT. Both T and DHT elicit sexual behavior in castrated male X. luevis (Kelley and Pfaff, 1976), and both androgens stimulate development of seminal vesicles in D. pictus (N’Diaye et al., 1974). It is not known whether ring A reduction of T is necessary for androgen action in anurans, as is the case for some androgen-responsive tissues of mammals. The presence of considerable 5~ reductase activity in the bullfrog is reminiscent of a similar situation in the immature rat (see Rivarolaet al., 1975). 5wReductase activity is low in the newborn rat, but greatly increases from about Day 15 to Day 30 or 40 (resulting in the formation of androstanediols and DHT), then declines at puberty and remains low in the adult; testosterone production decreases during the period of high Sa-reductase activity. Androstanediol and DHT are formed primarily in seminiferous tubules in the maturing rat and human, and high levels of these 5~ reduced androgens are correlated with the onset of meiosis or later stages of spermatogenesis. In the bullfrog, high DHT plasma levels and increased DHT/T ratios after in vitro testis incubation are observed in February-March, when late spermatogenic maturation stages are present, and the number of spermatozoa is increasing (Mullet-, 1976~). Likewise, in N. occider&is, Sa-reductase activity increases dramatically with the yearly onset of meiosis and sperm production (Gavaud, 1975, 1976). In the present results, Rana LH, but not FSH, increased the ratio DHT/T in vitro, suggesting LH dependence of testicular 5a-reductase activity in the bullfrog. In addition, hypophysectomy reduced, and hCG

118

CHARLES

injection increased, 5a+reductase activity in testes of another anuran, D. pictus (Ozon and Stocker, 1974). The control of testicular 5cY-reductase activity in mammals has received attention in view of its implications with spermatogenesis and puberty. Hypophysectomy greatly decreased 5~reductase activity in whole homogenates of immature rat testis, and injection of ovine LH restored this activity, but FSH or testosterone propionate injections were ineffective (Nayfeh et al ., 1975). Thus the presence (developmentally or seasonally) and gonadotropic control of testicular 5areductase activity are similar in some species of mammals and amphibians. Gonadotropic

Specificity

The bullfrog testis exhibits a high degree of specificity for LH over FSH in terms of in vitro stimulation of androgen secretion. These results support the conclusion that injections of Rana LH, but not FSH, stimulate major increases in plasma androgens in normal and hypophysectomized bullfrogs (Mullet-, 1977). Histological and histochemical studies of frog testicular interstitial cells and androgen-sensitive tissues provide indirect evidence for mammalian LH specificity of androgen production in vivo (see Lofts, 1974). Furthermore, the present in vitro results for male frogs are in accord with experiments demonstrating LH specificity of in vitro progesterone secretion by bullfrog ovarian tissues (Licht and Crews, 1976). In contrast, results of Wiebe (1970, 1972), using X. laevis, suggest that amphibian testicular steroidogenesis may be stimulated by either LH or FSH. These observations are not easily reconciled with the present results, but differences may reflect the use of high doses of heterologous hormones or the type of response studied. In particular, the present results indicate that misleading or erroneous conclusions would have been obtained had only high doses or relatively narrow dose ranges been

H. MULLER

employed (e.g., Fig. 2). In addition, RIA of only T would have provided results substantially different from those obtained after RIA of DHT or T + DHT. Altematively, male Xenopus may not exhibit the LH specificity observed for R. catesbeiana testicular androgen secretion, or for Xenopus ovulation. Nevertheless, direct evidence for LH specificity of testicular and ovarian steroid secretion using homologous gonadotropins is available for R. catesbeiana and for A. tigrinum (Licht and Crews, 1976; Muller, 1976~ and present results; see Licht et al., 1977). In the present results, Rana LH potency was 30-50 x NIH-ovine LH, but Rana LH is only 0.33 x NIH-ovine LH in another anuran assay system, the Xenopus ovulation test (Licht et al., 1977). In mammalian assay systems, the potency of Rana LH (and other nonmammalian gonadotropins) is only about 0.001 x NIH-ovine LH (Licht et al., 1976b; Papkoff et al., 1976a). This phenomenon of phylogenetic (species) specificity is presumably a reflection of structural changes of both hormone and receptor throughout evolution. Such alterations cannot be adequately studied at present, but one aspect of these changes, LWFSH specificity, has now been examined in each vertebrate class. This subject has recently been reviewed by Licht and co-workers (1977). The lack of LH/FSH specificity in reptiles does not necessarily reflect the ancestral tetrapod condition; rather, early tetrapods may have exhibited specificity (as suggested partly by the present results), which was lost in reptiles after the divergence of the therapsid-mammalian lineage. Alternatively, LH-specific stimulation of testicular androgen production may have evolved independently in Amphibia and Mammalia. ACKNOWLEDGMENTS 1 am indebted to Dr. Paul Licht (Department of Zoology, University of California, Berkeley) and to Drs. Harold Papkoff and Susan W. Farmer (Hormone Research Laboratory, University of California, San

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Francisco) for the preparation and provision of nonmammalian gonadotropins, and for valuable advice. I thank Drs. Paul Licht and Howard A. Bern (Department of Zoology and Cancer Research Laboratory. University of California, Berkeley) for critically reading the manuscript. Mr. Hugh Meakin provided technical assistance. This work was supported by NSF Grant BMS-75-16138 to P. Licht and H. Papkoff, while the author was supported by NIH Training Grant CA-05045 to the Cancer Research Laboratory, University of California, Berkeley.

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