Characterization of the testicular binding site for iodinated rat FHH in the turtle, Chrysemys picta

Camp. Biorhem. Physiol. Vol. 82A, No. 4, pp. 891-897, 1985

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mc 1985Pergamon Press Ltd

CHARACTERIZATION OF THE TESTICULAR BINDING SITE FOR IODINATED RAT FSH IN THE TURTLE, CHRYSEMYS PZCTA Department of Biology,

WKFRID DUBOIS* and IAN P. CALLARD? University, Boston, MA 02215, U.S.A.; Telephone:

Boston

(617) 353-2432

(Received 11 April 1985)

Abstract-l.

To correlate the morphological observations with the known gonadotropic activity of FSH in the turtle testis, studies of the binding of iodinated FSH were conducted. 2. These demonstrated the presence of gonadotropin-binding sites of high affinity (apparent Kd = IO-” M) for [‘251]rFSH in turtle testicular membrane preparations. 3. Although these sites did not bind iodinated human LH or avian LH, these hormones, as well as PMSG and FSH, were effective competitive inhibitors of the binding of the radioligand. 4. Binding of the radioligand to the testis was influenced by duration of incubation and temperature. 5. Binding activity was lost after incubation with proteolytic enzymes (trypsin, pronase) but not with DNAase, lipase, collagenase and neuraminidase. 6. The binding exhibited target organ specificity (no binding observed in brain, epididymis, lung, muscle and pancreas). 7. In addition, the number of binding sites varied according to the stage of spermatogenesis, being highest when the tubules contained spermatocytes and spermatids, intermediate when the tubules consisted to Sertoli cells and spermatogonia and lowest at spermiation.

INTRODUCTION

In mammals, gonadotropin regulation of testicular function is mediated by FSH and LH receptors on the plasma membrane of Sertoli (Abou-Issa and Reichert, 1976; Means et al., 1976) and Leydig cells (Dufau et al., 1973; Catt and Dufau, 1978), respectively. FSH acts on the seminiferous tubules to regulate spermatogenesis (Dorrington and Armstrong, 1979) and LH induces testosterone synthesis and secretion by Leydig cells (Hall et al., 1969; Rommerts and Brinkman, 1980). In reptiles, pituitary-testicular interactions are less well understood. Even though two gonadotropins sharing colligative and immunochemical characteristics of mammalian FSH and LH have been isolated from the pituitary of the turtles Chelydra serpentina (Licht and Papkoff, 1974) and Cheloniu mydus (Licht et al., 1976), in vivo and in vitro studies on a selected group of reptilian species suggest a lack of specificity in the steroidogenic and spermatogenic responses of the testis to either FSH or LH (Licht et al., 1977; Callard et al., 1978). In addition, specific binding sites for human FSH have been identified in the testis of turtles (Pseudemys scripta, Chrysemys picta, Chelonia mydas and Chelydra serpentina), lizards (Anolis carolinensis, Cnemidophorus tigris) and a snake (Thamnophis sirtalis) (Licht and Midgley, 1976; Licht and Midgley, 1977; Licht et al., 1977), but these sites have not been characterized according to seasonal variation and their target cells, tubular or interstitial, have yet to be identified.

For Chrysemys picta the reproductive cycle has been described (Gibbons, 1968) and plasma testosterone correlated with seasonal changes in testicular histology (Callard et al., 1976). This study is an attempt to characterize the binding site for rat FSH in the testis of the freshwater turtle, Chrysemys picta, using a crude plasma membrane preparation and iodinated rat FSH ([‘251]rFSH) as the radioligand. MATERIALS AND METHODS

Animals Male Chrysemys picta were trapped from a freshwater pond in Sudbury, Massachusetts, and kept outdoors in round plastic pools with concrete blocks to allow basking. They were fed raw fish ad libitum. From November to March, the animals were moved indoors, kept in the same pools and maintained on a 12-h (light/dark) photoperiod. Hormones and reagents Porcine FSH (NIH-FSH-P2, rat FSH (rFSH-I-3), bovine LH (NIH-LH-B-4) and pregnant mare serum gonadotropin (NIH-PMSG-2, 2200 Ii/m;) were generously provided by the Pituitarv Hormone Distribution Program (NIAMDD, Bethesda, MD). Avian LH (aLH-AEl) was kindly supplied by Dr Cohn Scanes (Rutgers University, NJ). The enzymes trypsin (type III, bovine pancreas) pronase, collagenase (type IV), lipase (type VII) and deoxyribonuclease (type I, bovine pancreas) were ordered from Sigma. Sodium Fisher Scientific, metabisulfite was obtained from chloramine-T from Eastman Kodak Company (Rochester, NY) and carrier free [‘251]Na (NEZ 033L) from New England Nuclear (Boston, MA).

Iodination rFSH-13 was iodinated by the chloramine-T method (Greenwood et al., 1963). The procedure used diverged slightly from the modification developed by Reichert and Bhalla (1974). In brief, to a conical microfuge tube contain-

*Present address: Department of Animal Science, University of Florida, Gainesville, FL 32611, U.S.A. tTo whom reprint requests should be addressed. 891

892

WILFRII) DUBOIS

ing 20 &I of 0.25 M phosphate (PO,) buffer, pH 7.6, were added in sequence 5 #l (1 mgjml) of the hormone, 10 ~1 (1 mCi) of i’sI-Na. followed by 10 pi (1 ma/ml) of fresh chloramine-T solution. The mixture was kept on-ice. After 20 s, 5091 of sodium meta-bisulfite (1.6 mgjml) was delivered into the tube and sufficient time allowed for complete quenching of the chloramine-T. Using a Pasteur pipette previously rinsed in bovine serum albumen (BSA) (5% in 0.05 M PO,), the reactant mixture was transferred to a 1 x 24-cm column of Sephadex GSO previously equilibrated with 5:; bovine serum albumen (BSA) and eluted with cold 0.05 M PO, buffer. One millilitre fractions were collected into 12 x 75-mm polystyrene tubes containing 100 I of 5% BSA. Five microlitre (~1) aliquots from each fraction were used to determine the radioactive profile of the chromatogram. The incorporation of lZsI into protein was estimated by trichloroacetic acid (TCA) precipitation. Thus S-pi aliquots from each fraction were transferred to a parallel set of 12 x 75 mm glass tubes containing 200 ~1 of BSA. After adding 1 ml of ZOn/, TCA, all tubes were kept on ice for 30min and subsequently centrifuged at 14OOg for 15min. The supernatant was then discarded and the resulting pellet counted in a Searle gamma counter operating at 65Y0 efficiency. Each fraction of the protein peak was tested for the capacity to bind to a standard testicular homogenate prepared as described below from animals captured in June. Those fractions showing maximum specific binding after incubation with excess membrane protein were pooled and diluted in buffer to lO”cpm/~l. Small aliquots of the [‘?‘I]rFSH were stored at - 70°C and used in subsequent studies. Specific activities were determined using the radioactivity emitted by the iodinated rFSH as described by Reichert and Bhalla (1976). The value of each preparation varied from 60 to 114 r_lCi/pg with maximum binding in the presence of excess testicular homogenate ranging from 15 to ZOO;,of the total count added to the incubation medium. The sp. act. of this tracer was slightly higher (1.42 times) than that reported by Kubokawa and Ishii (1984) using the same hormone preparation and the lactoperoxidase method of Myachi ef al. (1972). Avian LH (avLH) and human (hLH) were iodinated as described by Leidenberger and Reichert (1972). Five micrograms of protein were incubated, in the cold, with 1 mCi of [““I]Na and IOpg of chloramine-T for 30s. The steps followed for separating bound from free lZ51were as delinated above for FSH. The sp. act. of avLH was 6OpCi/pg and that of LH, 8OpCiing. Binding studies were done with rat testicuiar homogenate prepared as described for the turtle and incubated usine the conditions outlined under [iZSI]rFSH-binding assay. Preparation of the crude membrane fraction Animals were sacrificed by decapitation. The testes were removed and care was taken to dissect away the tunica albuginea and adhering epididymal fragments. All steps for preparing the crude membrane fraction were carried out on ice. Buffers were kept chilled (4’C). A small piece of testicular tissue (1 mmj) was fixed in’ a solution of 0.1 M cacodylate-buffered glutaraldehyde, pH 7.2 and processed for microscopy in order to identify the stage of testicular development. Using a pair of fine scissors the rest of the tissue was minced in a petri dish with cold buffer and transferred to a glass homogenizer. Testes were homogenized for 10-15 s in 40 mM tris buffer, pH 7.4 containing 5 mM MgClz with a ground-glass pestle and motor driven at low speed. The resulting homogenate was then filtered through a single layer of Nitex and centrifuged at 250g for 10min at 4°C. The pellet (G-250) was discarded and the supernatant centrifuged again at 30,OOOg for 30min. The final pellet was reconstituted in buffer and a IOO-~1 aliquot taken for protein determination as described by Lowry ef al. (1951) after solubilization in 0.1 N NaOH solution. The rest

and

IAN P. CALLARD

of the homogenate was then reconstituted and diluted to a final concentration of 5 mg/ml with the same buffer containing 0.25:/, BSA. When not used immediately, l.O-ml aliquots were stored at -70°C. Preparations kept on ice for as long as 20 h or stored at -70°C appeared to show no adverse effect on their ability to bind iodinated FSH maximally.

[ ‘J51]rFSHbinding assay The procedure used to measure the binding of iodinated rFSH to the crude turtle testicular membrane preparation was es~ntialiv that develoned bv Bhalla and Reichert ( 1974) and modified by Licht and Midgley (1976). All reactions were carried out in 12 x 75 mm polystyrene tubes on crushed ice. To each tube were added in sequence: buffer, unlabelled hormone radioligand and membrane preparation in a final volume of 0.2 ml. The buffer used consisted of 40 mM tris. pH 7.4 with 5 mM MgC& and 0.25% BSA. The assay mixture was then vortexed, covered and incubated in a Dubnoff metabolic shaker for up to 20 h at 22-25°C. No further increase in specific binding was seen after 16 h. The shaker was set at 100 oscillations/min. The reaction was terminated by adding to each test tube 1.0 ml of chilled water. Separation of free and bound radioligand was performed by centrifugation at 14006 for 30min in an IEC Centra-7r centrifuge set at 4°C. The supernatants were discarded and the pellets resuspended in 1.0 ml buffer, agitated using a Vortex mixer and centrifuged for an additional 30 min. A single wash removed 90% of the unbound radioligand. The resultant pellets were counted in a Searle gamma counter model 1190, operating at 654:j efficiency. In all these studies specific binding (N) was determined by addition to the incubation medium of a 100-fold molar excess of unlabelled pFSH prior to the addition of the tracer (Abou-Issa and Reichert, 1976). Specific binding is defined as (B - N)/T where B is the total counts bound, N is the counts in the tubes containing the excess unlabelled pFSH and 7’ is the total counts added to each tube. ‘

-

Determination of the equilibrium dissociation constant of the [ ‘-“I] rFSH binding site To determine the equilib~um dissociation constant (rC,), increasing amounts of [‘251]rFSH were added to duplicate aliquots of a fixed concentration of membrane protein and incubated for 18 h at 22-25°C in a final volume of 200 ~1. Non-specific binding was measured at each radioligand concentration. The saturation binding data thus obtained was transformed into a Scatchard plot (Scatchard, 1949). Tissue speci~city ctf [ “-‘cl]rFSH binding The tissue specificity of [“%]rFSH binding was determined for testis, liver, brain, pancreas, muscle and epipidymis. Homogenates were prepared as described above for the testicular CMP. Seventy-five micrograms of membrane protein for the respective tissue of the same animal were each incubated in triplicate with a saturating concentration of radioligand in a final volume of 0.2mI. Nonspecific binding was determined in duplicate in a matching set of tubes. The effect of enzymes on the integrity of the testicular

[ ‘2Sl]rFSH binding site Digestion of the testicular tissue by trypsin (50units). nronase (5 g), collanenase (5 g), linase (5 g) and DNAase (IO0 units) was carhed out at 37°C for 30 min. Enzyme solutions prepared as described by the distributor were added to 200 g protein of the CMP. The reactions were stopped by adding excess cold buffer. After centrifugation the pellets were washed in I ml buffer, centrifuged again and incubated with [‘Y]rFSH. Other samples incubated under identical conditions but in the absence of enzymes served as controls. Specific FSH binding was determined in triplicate for the enzyme-treated tissue.

Testicular

binding

of FSH

l$fecf of other protein hormones on [12’I]rFSH binding

Table I. Seasonal changes in testicular otroein receotors

The ability of other hormones to interfere with binding of the radioligand was evaluated by comparing the capacity of porcine FSH (PZ), avian LH (AE-l), PMSG, bLH (4) growth hormone (S9) and prolactin (B2) to compete for [‘Z51]rFSH-testicular binding sites.

Stage

Slutistical analysis Changes in receptor level during the seasonal cycle were analysed by Duncan’s multiple range test (Sokal and Rohlf, 1969).

Protein (fmole/mg) (SEM)

I

107.70 191.33 150.97 67.16

2 3 4

Seasonal changes in testicular gonadotropin receptors Seasonal changes in testicular gonadotropin receptors were determined by Scatchard analysis using either fresh tissue samples or samples frozen on dry ice and stored at - 7o’C. When frozen samples were used, they were thawed on ice prior to homogenization. To measure possible changes in gonadotropin receptors, increasing doses of iodinated rFSH were added to a series of tubes each containing a constant concentration of testicular CMP prepared from animals trapped in Sudbury, Massachusetts, from April to September. November and December samples were obtained from a supplier (Nasco) or from Sudbury animals maintained indoors on a 12-h (light/dark) photoperiod. In addition, pieces of tissue were routinely processed for light microscopy to ascertain the stage of tubular development.

893

(15.13) (14.04) (19.68) (16.76)

gonadn 3 3 8 3

SD, standard deviation. Analysis of variance shows that there is a dilTerence between the means (F= 29.0, d[= 3.13, P ~0.05). Duncan’s multiple range test indicates that the four means arc significantly different from each other (P < 0.05). Table 2. Binding

of iodinated rFSH, hLH and avLH to rat and turtle testis

Protein (pg)

[‘2SI)rFSH

Specific binding* [“SIlavLH I”SIlhLH

100 100

IS”;, Not detected 16:; 5”’ 0.8:, 6.2Y” 10 *Values represent “/, of total (25,000 cpm) counts added. n = 2.

Turtle Rat

contrast, no significant specific binding could be demonstrated in the brain, epididymis, liver, muscle and pancreas. Effect of enzyme digestion on the integrity testicular [ “‘I]rFSH receptor

RESULTS

of the

Specific binding of FSH to the testis was saturable (Fig. 1). Scatchard analysis (Fig. lb) was consistent with the presence of a single class of binding sites with an average equilibrium dissociation constant 2.23 x IO-” M. The binding capacity varied depending on the stage of tubular development (Table 1). No significant binding of “‘FhLH of [“‘I]avLH was observed (Table 2).

The effect of trypsin, DNAase, lipase and collagenase are presented in Fig. 3. Specific binding was reduced to 8”/, of control samples after pretreatment of the receptor preparation with trypsin or pronase. In contrast, collagenase, DNAase and lipase had virtually no adverse effect since specific binding was close to values obtained with control samples incubated without enzymes. The figure also indicates that preincubation at 36°C in the absence of radioligand resulted in a 45% loss of binding capacity.

Tissue speciJicity of [ ‘*‘I]rFSH binding

Specificity

The amount of specifically bound radioligand was highest (11&120fmole) in the testis (Fig. 2). In

Specific binding of iodinated rFSH to turtle testis was inhibited in a dose-dependent manner by pFSH,

Determination

of the K, of the [ ‘*‘I]rFSH binding site

of the receptor preparation for [ I*-‘l]rFSH

‘\ Kdr2.2

TOTAL ’ 251-rFSH

64)

x IO-” M (7)

BOUND (PM)

Fig. 1. (a) Saturability of the testicular rFSH-binding site. Increasing doses of iodinated rat FSH were added to a fixed concentration of membrane protein obtained from animals sacrificed in November (stage 4). Total (T), specific (S) and non-specific (N) binding are shown. Each point represents the mean of duplicate determinations from two animals. (b) Scatchard analysis of the saturation binding in (a) used to estimate equilibrium dissociations constant (Kd) and density of available [“‘I]rFSH receptors,

894

WILFRIDDUBOISand

T

B

E

L

M

IAN

P. CALLARD

P

Fig. 2. Tissue specificity of [‘z51]rFSH binding. Specifically bound radioligand in testis (T), brain (B), epididymis (E), liver (L), muscle (M) and pancreas (P) expressed in fmolejmg protein of homogenate. Each bar represents the mean for duplicate samples.

-01

.I

1

10

x103

Ng LlnlabeiedHormone Fig. 4. Specificity of the receptor for the radioligand. Competitive inhibition of [‘251]rFSH binding by increasing concentration of FSH-P,, aLH, bLH or PSMG. No inhibition could be obtained with growth hormone (CH) or prolactin (PRL). Each point represents the mean of duplicate samples.

DISCUSSlON

Cn

No

D

L

T

k

C

P

Fig. 3. The effect of enzyme digestion on the integrity of turtle testicular rFSH receptors. Specific binding of [“51]rFSH to receptors after preincubating the crude membrane prepa~dtion at 37°C alone (No) or with collagenase (C). DNAase (D), Lipase (L), Pronase (P) and trypsin (T). Each bar represents the mean of triplicate sampies.

aLH, bLH and PMSG

but not by growth hormone

or prolactin (Fig. 4). Semonat change.? in testicular rFSH receptors

The amount of specifically bound [‘“‘l]rFSH varied with the stage of spermatogenesis. Four stages were observed: (1) characterized by spermatogonia and Sertoli cells containing lipid droplets (April-May); (2) characterized by mitotic figures and spermatocytes at the iuminal margin (June); (3) characterized by early and late stage spermatids at the luminal margin (July-September); and (4) spermiation (September-November). Receptor density was highest during Stages 2 and 3, lowest during Stage 4 and intermediate during Stage 1. Specific binding was 191, 151, 67 and 107 fmoles/mg protein at each of the respective stages (Table 1). Analysis of variance shows that there is a difference between the means (F = 29.0, df = 3.13, P < 0.05) and the four means are significantly different from each other as indicated by Duncan’s multiple range test (P < 0.05). There was no apparent change in the apparent & of the receptors. Thus the changes observed appear to reflect changes in the number of available binding sites.

We have demonstrated that the testis of Chrysemys high-affinity binding sites for rFSH. These sites vary in number with the stage of the testicular cycle and do not bind iodinated hLH or avLH. The highest number of binding sites, as indicated by radioligand binding, was found in the testis where the hormone is known to stimulate steroid synthesis (Cahard and Ryan, 1977; Lance et al., 1977). Nontarget organs such as the brain, liver, pancreas, muscle and epididymis exhibited practically zero uptake or less than 10% of the binding capacity of the testis. In an attempt to further identify the membrane component(s) involved in the radioligand binding-site interaction, the binding capacity of the homogenate was evaluated after incubation treatment with enzymes which specifically hydrolyse DNA, lipids, proteins or collagen. That the radioiigand is in fact binding to a protein component of the membrane is indicated by the susceptibility of the testicular CMP to trypsin and pronase and its resistance to DNAase and lipase digestion. Even though a trypsin inhibitor was not used after the trypsin-treated tissue was washed and then incubated with [1251]rFSH,the absence of binding may not be explained by the action of residual trypsin on the iodinated ligand itself because when [“‘I]rFSH incubated with trypsinized liver, brain, pancreas or muscle was recovered by centrifugation and incubated with intact testicular tissue, specific binding was not significantly affected. (Data not shown.) In this study we have used ]‘251]rFSH as a tracer because Callard and Ryan (1977) demonstrated that this preparation was more potent than hFSH or oFSH in stimulating testosterone synthesis in enzyme-dispersed testicular cells from Chrysemys. A radioligand of high specific activity was desirable in order to carry out saturation-binding experiments picta contains

Testicular

binding

over a wide range of [‘2SI]rFSH concentrations. There is no reason to believe that the binding capacity of [‘*‘I]rFSH was significantly impaired since, depending on the stage of testicular development, specific binding varied from 5 to 20”/, of the total counts added. This range encompasses the values (6-10x) reported for [“51]rFSH binding to the turtle Geoclemys reevesi (Kubokawa and Ishii, 1984) and [‘*‘I]hFSH to the turtle Chrysemvs picta (Licht and Midgley, 1976) using crude testicular membranes prepared in an identical manner. Licht and Midgely (1976) were the first to show binding of a mammalian gonadotropin (hFSH) to Chrysemys testis. However, their study did not include all stages of the testicular cycle and was limited to demonstrating: (1) the dependence of this binding on time and temperature and (2) its inhibition by hFSH and oFSH, but not by oPRL and BGH. In the present study, we have consistently observed inhibition of [‘*‘I]rFSH by bLH and PMSG. Even at concentrations of 5pg, Licht and Midgely (1976) observed no appreciable displacement of [‘*‘I]hFSH binding by hCG or oLH, 1Opg of bLH decreasing binding only to 80% of control. The differences observed on the effect of bLH on mammalian FSH binding to turtle testicular tissue seen in this study and that of Licht and Midgley can be attributed to the following: (1) that we have used rFSH instead of hFSH, (2) the binding-inhibition studies were done using tissue at different stages of the testicular cycle, (3) the use of different LH preparations, i.e. Pierce vs NIH and (4) contamination of the other hormones with FSH. The ability of bLH to inhibit [‘*‘I]rFSH binding, like the inability of [‘251]avLH to bind to testicular tissue, are not consistent with the effect of these hormones on steroid synthesis (for review see Licht et al., 1977). On one hand mammalian LHs do not have significant intrinsic stimulatory activity on steroid synthesis by Chrysemys testis in vivo (Callard et al., 1976; Lance et al., 1977) or in vitro (Callard and Ryan, 1977). On the other hand avLH induces an increase in testosterone in enzyme-dispersed testicular cells and inhibits [‘*‘I]rFSH binding to Chrysemys testicular tissue (Callard and Ryan, 1977). The effect of PMSG on rFSH binding is consistent with its ability to induce androgen synthesis in vitro (Callard and Ryan, 1977), although to a lesser extent than rFSH, hFSH, oFSH or avLH. The inability of [‘*‘I]avLH to bind may be a consequence of damage caused by the iodination procedure to a portion of the avLH molecule involved in binding. However, it is also possible that the optimum conditions for avLH binding may be different from those for FSH. In this respect it is interesting to note that maximum steroid secretion and spermatogenesis occur under diferent environmental conditions. In this study we did not attempt to optimize or identify the optimum conditions for avLH binding. Hormones exert their action by binding to specific high-affinity receptors on target organs. High affinity is reflected in a low dissociation rate constant (&) (Roth, 1973; Hollenberg and Cuatrecassas, 1975). In mammals, irrespective of the source of tissue (bovine, ovine or rodent), tracer (homologous or heterologous) or extent of purity of the receptor preparation

of FSH

895

Table 3. Affinity and capacity of receptors species SOUICC

Turtle Turtle Rat Rat’ Calf? Pig Quail Fowl Sparrow Turkey Chicken

&

2.2 x 5x 6.7 x 7.0 x 4.7 x 5.6 x 4.1 x 1.5 x 8.6 x 3.3 x 1.6 x

00

lo-‘0 10-g 10-10 10-I’ lo-‘0 IO-” 10-P 10-g IO-” 10-g IO-”

from various

vertebrate

Reference Dubois and Callard Kubokawa and Ishi (1984) Bhalla and Reichert (1974) Abou-Issa and Reichert (1976) Abou-Issa and Reichert (1977) Maghuin-Rogister ef nl. (1978) Tsutsui and Ishil (1978) Ishii and Adachi (1977) Ishii and Farner (1976) Bonna-gallo and Licht (1979) Bonna-Gallo and Licht (1979)

*Purified membranes. tDetergent solubilized.

(whole testis homogenate, crude membrane preparations or detergent solubilized membranes), reported Kd values for FSH-binding site in the testis range from 10m9 to lo-” M (Table 3) and Scatchard analysis of binding data often suggests the presence of two receptor populations with different affinities for the ligand. Similar observations have been made in avian species (Ishii and Farner, 1976; Ishii and Adachi, 1977; Tsutsui and Ishii, 1978, 1980; Bonna-Gallo and Licht, 1979) using Scatchard analysis of binding data obtained from saturation analysis (fixed amount of tissue incubated with increasing concentration of tracer) or competitive inhibition data (constant radioligand and tissue incubated with increasing amount of unlabeled hormone). In this study the binding site for [‘*‘I]rFSH has a mean Kd value of 2.2 x lo-” and the Scatchard plot was linear. These observations are in agreement with those made by Kubokawa and Ishii (1984) who reported Kd values ranging from 10d9 to 10 lo M and linear Scatchard plots for [‘*‘I]rFSH binding sites in the testis of the turtle Geoclemys reevesi. These studies suggest that the testes of Chrysemys and Geoclemys contain a single type of high-affinity binding sites for rFSH. By applying [‘2SI]rFSH on frozen tissue sections Licht and Midgely (1977) demonstrated the presence of hFSH-binding sites in the testis of two lizards (Anolis caroh’nensis and Cnemidophorus tigris) and a snake (Thamnophis sirtalis). Even though no data were shown for male Chrysemys, the authors indicated that reduced silver grains were distributed over regions containing peripheral Sertoli cells and spermatogonia. Orth and Christensen (1977) have discussed the limited resolution of topical autoradiography so well illustrated by Desjardins et al. (1974). Even in the rat where Leydig cells form distinct islands between seminiferous tubules this procedure has yielded conflicting results. In Chrysemys where Leydig cells are closely apposed to the seminiferous tubules (Callard et al., 1976) results from topical autoradiography may not be conclusive. With this in mind we attempted but failed to observe reduced silver grains in the tubular or interstitial compartments of the testis by following the procedure described by Orth and Christensen (1977). Likewise we could not confirm Licht and Midgley’s (1977) observations for the turtle using their procedure (topical autoradiography) and testicular tissue

896

W~LFRIU DUBOIS and

at two different stages of the testicular cycle. Thus to date we are unaware of any data illustrating the localization of specific binding sites for any g&adotropins (homologous or heterologous) in the turtle by using autoradiography. In summary, the testis of the freshwater turtle Chrysemys pictu contains binding sites for [‘ZSI]rFSH but not for [‘*‘I]hLH or [‘?]avLH. These sites have a high affinity for the ligand and change in number with the stage of the testicular cycle. The exact location of these sites, whether tubular or interstitial, and their specific target cells, whether Sertoli. Leydig or germinal, remains unresolved. Acknowledgement-This 78-08201 and 81-04144

work was supported to I. P. Callard.

by NSF PCM

REFERENCES

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IAN P. CALLARU

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