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BACKGROUND: Puberty in male Atlantic salmon in aquaculture can start as early as after the first winter in seawater, stunts growth and entails welfare problems due to the maturation-associated loss of osmoregulation capacity in seawater. A better understanding of the regulation of puberty is the basis for developing improved cultivation approaches that avoid these problems. Our aim here was to identify morphological and molecular markers signaling the initiation of, and potential involvement in, testis maturation. METHODS: In the first experiment, we monitored for the first time in large Atlantic salmon males several reproductive parameters during 17 months including the first reproductive cycle. Since testicular growth accelerated after the Winter solstice, we focused in the second experiment on the 5 months following the winter solstice, exposing fish from February 1 onwards to the natural photoperiod (NL) or to continuous additional light (LL). RESULTS: In the first experiment, testis weight, plasma androgens and pituitary gonadotropin transcript levels increased with the appearance of type B spermatogonia in the testis, but testicular transcript levels for gonadotropin or androgen receptors did not change while being clearly detectable. In the second experiment, all males kept under NL had been recruited into puberty until June. However, recruitment into puberty was blocked in ~ 40% of the males exposed to LL. The first morphological sign of recruitment was an increased proliferation activity of single spermatogonia and Sertoli cells. Irrespective of the photoperiod, this early sign of testis maturation was accompanied by elevated pituitary gnrhr4 and fshb and testicular igf3 transcript levels as well as increased plasma androgen levels. The transition into puberty occurred again with stable testicular gonadotropin and androgen receptor transcript levels. CONCLUSIONS: The sensitivity to reproductive hormones is already established before puberty starts and up-regulation of testicular hormone receptor expression is not required to facilitate entry into puberty. The increased availability of receptor ligands, on the other hand, may result from an up-regulation of pituitary Gnrh receptor expression, eventually activating testicular growth factor and sex steroid release and driving germ and Sertoli cell proliferation and differentiation. Erratum in Correction to: Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

LH and hCG are members of the glycoprotein hormone family, which consists of noncovalently associated α- and β-subunits (1). Upon dissociation of α-β dimers, each subunit loses high-affinity hormonal activities. LH and CG interact with the LH/CG receptor. This is expressed in the gonads of both sexes at specific stages of development and, particularly, in the ovary during the ovulation cycle (2, 3).

The pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), play important roles in regulating the steroidogenic and gametogenic functions of the gonads. The actions of these hormones are mediated by specific cell-surface receptors, the LH/hCG receptor (LH/hCGR), and the FSH receptor (FSH-R). Cloned cDNAs for these receptors were recently isolated in several laboratories (1–4). The deduced amino acid sequences of these two receptors predicts 7 potential transmembrane domains, indicating that they are related to the G-protein-coupled receptor family.

A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic α-subunit peptide (α26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of α-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor.

5α-Androstane-3β,17β-diol (3β-diol) was observed to inhibit [3H]estradiol ([3H]E2) binding to testicular cytosol preparations from both immature and mature rats with a potency of 1% that of diethylstilbestrol. The nature of the inhibition of [3H]E2 binding by 3β-diol was shown to be competitive with a KI of approximately 12 nM. This high competitive potency for a C19 steroid required both 3β- and 17-hydroxyl groups, and a reduced C5 position to inhibit E2 binding. Despite the interaction of 3β-diol with E2 receptor in vitro, injection of immature rats with 25 or 250 μg 3β-diol 1-6 h before autopsy did not occupy or deplete the testicular cytoplasmic E2 receptor. When administered to immature female rats, 3β-diol did not show estrogenic agonistic or antagonistic activity in the uterus. These results suggest that the interaction of 3β-diol with the testicular receptor may be of little physiological significance.

Testicular and prostatic androgen-receptor complexes as well as uterine estradiol-receptor complexes, partially purified by ammonium sulfate precipitation (15–37%), were bound to germ cell chromatin. At equivalent concentrations, less testicular androgen-receptor complexes bound to chromatin than did the other two steroid-receptor complexes. Addition of a partially purified testicular androgen-receptor preparation with prostatic androgen-reeeptor or uterine estradiol-receptor preparation to the binding interaction mixture reduced the binding of either of the latter two steroid-receptor complexes to chromatin. These data suggest the presence of inhibitory factor(s) in the testicular receptor preparations. Testicular cytosols were fractionated by ammonium sulfate precipitation into fractions A (15–37% saturation), B (37–50%) and C (50–75%). All fractions inhibit binding of these steroid-receptor complexes to chromatin. Fractions A and B appear to be heat labile, while fraction C was more stable. Further fractionation of A and C fractions on DEAE cellulose yielded A1 and C1 (filtrates) as well as A2 and C2 (0.3 M Nacl eluents), respectively. Subfractions A1, A2, and C1 contained inhibitory factors for the binding of steroid-receptor complexes to chromatin while C1 showed no effect. These data demonstrated that testicular cytosol contains a variety of inhibitory factors which affect the binding of both androgen-receptor and estradiol-receptor complexes to chromatin.

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.

By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

Androgen receptor concentration was measured by exchange with 3H-dimethylnortestosterone (DMNT) in cytosol and nuclear extracts from testes of rats 15-90 days of age. Dissociation kinetics verified the necessity of an extended incubation (86 h) for maximum exchange at 4 degrees C. Nuclear androgen receptor concentration per mg DNA decreased between 15 and 25 days of age, from 375 to 146 fmol per mg DNA, then increased to 584 fmol per mg DNA at 90 days. Testicular receptor content also increased between 25 and 90 days of age. Cytosol receptor concentration patterns were similar to nuclear androgen receptor patterns. The affinity of the receptor for the ligand did not change with age (mean Kd = 0.88 nM). No significant difference in androgen receptor concentration per cell was detected between cultured peritubular cells from animals 25 and 45 days of age. Androgen receptor concentrations in freshly isolated peritubular cells could not be determined. There also was no difference in receptor concentration per cell in a Leydig cell-enriched fraction from animals between 25 and 45 days of age. Although androgen receptor concentrations per Sertoli cell increased between 15 and 35 days of age, the increase in Leydig cell number over the same period probably accounted for approximately 75% of the increase in receptor per testis between 25 and 45 days of age.

SUMMARY A modified bovine testicular receptor was used to evaluate highly purified follicle-stimulating hormone (FSH) from a number of species. The particulate receptor obtained from adult bovine testes could be stored frozen or lyophilized for long periods without appreciable decrease in the binding of the ligand facility or in loss of specificity. The bovine testis receptor binds twice as much 125I-labelled ovine FSH as 125I-labelled human hormone. When FSH from different species was compared against NIH-FSH-S10, using various FSH ligands, the ovine hormone was clearly the most active, although many had comparable in-vivo biological potencies. The results suggest that there is probably some species specificity in the hormone–receptor interactions. As the ovine hormone is structurally closer to the bovine, from which the receptor was derived, it appears to have the highest activity in vitro. Marked differences in the biological activities of the different preparations between the human chorionic gonadotrophin-augmentation test and the in-vitro assays have been observed. In the in-vitro assays, all preparations, with the exception of the porcine hormone preparation, were less active and the ratio of bioassay/radioreceptor assay varied widely. In the radioreceptor assays, all FSH preparations except pregnant mare serum gonadotrophin (PMSG) showed parallel inhibition curves. The three different PMSG preparations examined gave inhibition lines that were parallel to each other.

We have studied the effects of adrenalectomy and glucocorticoid injections on insulin binding in membranes from rat testis and liver. Glucocorticoids were administered for 7 days to adrenalectomized rats at daily doses of 30 or 300 μg for dexamethasone and 100 or 1000 μg for prednisolone. Glucocorticoids, at the selected doses, were associated with 5- to 10-fold increases in basal insulin levels with no significant changes in glucose concentrations. As previously shown by other studies, down-regulation of insulin receptors was observed in liver membrane particularly at the higher dose of steroids. Such an effect was not found in the testis. By contrast, the number of high-affinity sites in the testis was slightly increased with the higher doses of dexamethasone and prednisolone. However, percent 125I-labelled insulin binding was not significantly changed after corticotherapy. These results are in accord with our previous studies and suggest that the testicular receptor for insulin is not affected by mild to moderate changes of insulin concentrations, but that it can be modulated by glucocorticoids through other mechanisms.

Injection of male rats with 40-200 ng of [D-Leu6, des-Gly-NH2(10)]LHRH ethylamide for 7 days caused a maximal 80% reduction of testicular LH/hCG receptor level with one injection per day being as efficient as 3 daily injections. A similar inhibitory effect was observed on testicular PRL receptors. Testis and seminal vesicle weight as well as plasma testosterone levels were also significantly reduced by this treatment. These data indicate that a LHRH agonist, when given at a relatively low dose, is capable of reducing testicular LH/hCG and PRL receptor levels as well as testicular function, the effect being probably mediated by increased endogenous gonadotropin secretion.

Receptors for [125I]hCG were found in adult human testis. The specific binding of [125I]hCG to testicular receptor is temperature dependent and is a saturable process with respect to added receptor protein and hormone. Scatchard analysis revealed a dissociation constant of 5.0 X 10(-10) M, and 6.2 fmol binding site/mg protein. Intact unlabeled hCG effectively inhibits the specific binding of [125I]hCG to human testicular receptors. For inhibition of binding of [125I]hCG, the alpha subunit has 3.0% of the potency of intact hCG and the beta subunit has 0.4% of the potency of intact hCG. Specific binding is pH dependent, with an optimum at pH 7.4. Brief exposure to extremes of pH causes irreversible damage to the receptors. Incubation with protease and trypsin results in an almost complete loss of binding activity, while ribonuclease, deoxyribonuclease, phospholipase C, or neuraminidase treatment does not significantly alter hormone-binding activity. Binding activity was found to be positively correlated to the concentration of intratesticular testosterone.

Two low-molecular-weight gonadal peptides, IRRP3 and IRRP5, have been isolated from sheep ovaries having different biological properties in male rats. The peptide IRRP3 has specific effect on FSH secretion as well as release at the pituitary level, in vitro binding of labeled FSH to testicular receptor, and in vivo uptake of labeled FSH by the testis, whereas peptide IRRP5 has specific effect on LH secretion at the pituitary level, in vitro binding of labeled LH to testicular receptor, and in vivo uptake of labeled LH by the testis.

In vivo administration of gonadotropin-releasinghormone (GnRH) inhibits testosterone production in intact rats and increases testicular GnRH receptors. To delineate the relationship of these effects and to determine whether GnRH induced its testicular receptor by an extrapituitary mechanism, GnRH and LH receptors were studied in intact or hypophysectomized (H) rats. Adult, H, or sham-operated (S) males were injected with GnRH (6.7 μg every 8 h for 1, 2, 4, and 6 days). Some H rats were also treated with GH and PRL (150 μg/day) to maintain LH receptors. Decapsulated testes were assayed for GnRH and LH receptors or incubated to assess testosterone production in the presence or absence of hCG. GnRH treatment in S rats resulted in a biphasic change in GnRH receptor numbers. The basal receptor concentration (212 ± 27 fmol /mg protein) increased to a peak at 2 days (501 ± 32 fmol/mg) before declining to control values after 6 days of treatment. GnRH receptors increased 2-fold 1 day after hypophysectomy (449 ± 5...

A simple radioreceptor-assay technique for human follicle stimulating hormone (hFSH) with use of rat testicular slice and enzymatically 125I-labeled hFSH was described. It was shown that 125I-hFSH bound to the testicular slice (30mg) was approximately 2% of the total amount added to the tube (2ng/tube), half of which was displaceable in the presence of 1μg/tube of unlabeled hFSH. Although only small amount of 125I-hFSH was bound specifically to the tissue, the binding was not inhibited by other peptide hormones tested. The testicular receptor for hFSH had high affinity constant (2.5×109M-1) and hormonal specificity, suggesting a possibility of the physiological application of this radioreceptor assay for the measurement of hFSH in body fluids.

It has long been recognized that for a hormone to activate a target tissue, it must bind to some element of the cell. Roth and his colleagues (1) and Goodfriend and Lin (2) introduced appropriate techniques for directly studying the binding of a hormone to its target cell. Catt, Dufau and their colleagues (3, 4) and Reichert et al. (5) have characterized the binding of labeled human Luteinizing Hormone (hLH) and of human Chorionic Gonadotropin (hCG) to rat testis in vitro and impressive advances have been made in the isolation of the specific receptor hCG.

A testicular site of action for LHRH was first indicated by observations that LHRH agonists bound to high-affinity receptors in the interstitial tissue of the adult rat testis[1–5] and inhibited Leydig cell function in hypophysectomized rats in vivo [6–8]. Although there have been several reports since of proteins or peptides in testicular tissue and seminal plasma that have “LHRH-like” properties [9–24], the biochemical characteristics of most of these molecules, their relationships to the mammalian hypothalamic LHRH decapeptide and to one another, and their physiological significance remain poorly understood.

In in vitro studies, the synthetic estrogens diethylstilbestrol and diethylstilbestrol sodium phosphate inhibited the binding of 125 I ovine lutropin to the rat ovarian receptor and 125 I ovine follitropin to the bovine testicular receptor. As the lutropin binding to receptor is affected to a greater extent, a preferential inhibitory effect may be suggested. Removal of the estrogens from the incubation medium by washing does not restore gonadotropin binding ability, indicating a strong effect. The two compounds were effective in displacing the labeled gonadotropin from the preformed receptor-hormone complex. This effect increased with time of incubation. It appears unlikely that the interference of gonadotropin-receptor interaction may be because of increased hormone and/or receptor degradation by the two compounds.

Discovery of LHRH1,2 and the availability of its potent agonistic analogues has led to a rapid increase in our knowledge of the pituitary-gonadal axis and has offered new and exciting therapeutic approaches in reproductive endocrinology as well as in sex steroid-dependent diseases.

The interaction of 125I-labelled hFSH with primate testicular tissue from 4 species of adult monkeys (Macaca mulatta, M. nemestrina, M. fascicularis and Papio cynocephalus) was investigated. 125I-labelled hFSH binding to a particulate fraction (P1, 40 000 g) of frozen testes was highly specific and saturable. Displacement curves generated using the P1 fraction of testes from the 4 species and 125I-labelled hFSH and unlabelled FSH were very similar. The binding of FSH to the monkey testicular receptor was not species specific because purified FSH from heterologous species such as horse, sheep, pig and rat were very effective in competing with 125I-labelled hFSH for binding. The equine FSH was about 10 times more active than hFSH in this respect. Similarly, 125I-labelled ovine FSH bound as well as labelled hFSH to the testes fractions of all 4 monkey species. In marked contrast to the high binding of 125I-labelled hFSH, binding of 125I-labelled hCG with rhesus monkey testis homogenates and P1 fractions was very low. The FSH receptor in the adult rhesus monkey testis was present in much larger quantity than the LH receptor and was more readily detectable. Our studies show that frozen primate testis can be utilized for investigating testicular-FSH interactions.

Stimulated by the unexplained lack of success of LHRH and its agonists in the treatment of infertility in men1, we investigated in detail the effect of acute and chronic administration of these peptides on testicular functions in experimental animals. We then made the observation that short-term administration of an LHRH agonist to adult male rats leads to a loss of testicular LH and prolactin receptors, as well as to decreased serum testosterone levels accompanied by inhibition of ventral prostate, seminal vesicle and testis weight2–5. It is of great interest that, among the species so far studied, man is the most sensitive to the inhibitory effect of treatment with LHRH agonists on testicular steroidogenesis5,6. In fact, while, in the rat, treatment with LHRH agonists increases 5α-reductase activity and formation of 3α-androstanediol as well as 5α-dihydrotestosterone, which can partially counteract the inhibitory effect on testosterone production5,7, no such effect is seen in man, where androgen biosynthesis can be completely inhibited and medical castration is thus achieved relatively easily with no secondary effect other than those related to low circulating androgen levels5,8,9.

The effects of both high and low molecular weight inhibin preparations on testicular and pituitary receptors were studied. Both these preparations were able to inhibit the binding of labelled hFSH to testicular receptor in a dose related manner, but were unable to affect the binding of labelled hCG to its receptor, suggesting that the observed inhibition of FSH binding was specific to inhibin. In addition, the binding of LHRH to pituitary receptors was affected by inhibin preparations. Interestingly, the antiserum raised against high molecular weight inhibin was able to neutralize, totally, the biological effect of high molecular weight inhibin, and only, partially, the biological effect of low molecular weight inhibin.

A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 ×10-11M, and 5.9 × 10-14mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40°C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5 μg/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone.

In the present study we have examined the developmental changes in the concentration of receptors for calcitriol in high-salt cytosol from the rat testis. Receptors for calcitriol were undetectable (less than 0.4 fmol/mg protein) until day 24, after which there was a rapid increase to reach adult levels (6-8 fmol/mg protein) between day 50-60. The lack of receptors in high-salt cytosol from the immature rat testis is not due to degrading enzymes, since cytosols prepared from the combination of equal volumes of testis homogenates from immature and adult rats had binding levels exactly half of that found in "adult controls". Furthermore, the increase in specific binding of [3H]calcitriol during development is due to an increase in the number of receptor sites, and is not due to a change in the apparent affinity of the receptors (Kd approximately equal to 1 X 10(-11) M at 0 degrees C). These results may explain why we previously were unable to demonstrate calcitriol receptors in cultured Sertoli cells and peritubular cells isolated from 19-day old rats. Furthermore, they indicate that calcitriol may be of minor importance for testicular function in the immature rat. The role of calcitriol in the pubertal and adult testis remains to be established.

The isolation, identification, synthesis, and biologic evaluation of the hypothalamic peptide, luteinizing hormone (LH) releasing hormone (LHRH), have provided a framework for the potential use of this natural substance in the treatment of infertility (Schally et al., 1976, 1978; Vale et al., 1977; Corbin and Beattie, 1976). With the further extensive syn-thetic development of agonistic derivatives of LHRH (super analogues), and the pharmacologic elucidation of their highly potent LH-releasing and ovulation-inducing properties in laboratory animals, it was envisioned that such peptidic agents would be most useful in treating female and male fertility disorders. Further testing revealed that such compounds, in spite of their putative profertility classification, were paradoxically antifertility in nature. This is evidenced by the ability of such agonists to terminate pregnancy postcoitally, reduce fecundity precoitally, and retard female puberty in laboratory animals; in humans and farm animals, they generally result in luteal incompetence despite their intended use for ovulation induction and establishment of a viable pregnancy (Corbin et al., 1978).

As an initial approach to the study of seminiferous tubule androgen receptors in disordered spermatogenesis, cytosol androgen receptors were studied in rats with experimental cryptorchidism. Two weeks after the testis had been repositioned in the abdomen of 6-week-old rats, the animals were hypophysectomized to deplete the testis of androgen, and 1 week later they were killed. Androgen receptor binding was studied in seminiferous tubule cytosol using [3H]methyltrienolone as the radiolabelled probe. The androgen-binding capacity of cryptorchid testis, when expressed as fmol bound/testis, was reduced to 50% of control, in parallel with the decline in testis weight. No change in binding affinity was found. Sucrose density gradient centrifugation using a vertical tube rotor revealed a 9S molybdate-stabilized receptor under low-salt conditions in both cryptorchid and scrotal seminiferous tubule cytosol. Receptor-complex stability studies, analysis by gel filtration and DEAEcellulose chromatography produced similar results in cryptorchid and scrotal tubules. The mechanism for the reduction in testicular receptor content of an abdominal testis remains to be clarified. The demonstration that testicular androgen receptors can be reduced by cryptorchidism suggests that further studies may indicate the role of receptor binding in testicular function. J. Endocr. (1986) 109, 427–433

The gametogenic and steroidogenic functions of the ovary and testis are both well known and extensively studied in a number of species including primates. Besides sustaining the growth and secretory activity of the accessory sex organs, the gonadal steroids also exert important positive and negative feedback effects on pituitary gonadotropin secretion in both sexes. The suggestion that the testis (gonads) may also secrete additional hormonal factors that are soluble in aqueous solutions and different from steroids, was made a long time ago. It was further suggested that this factor(s) may be involved in the regulation of pituitary gonadotropin secretion. The name inhibin was thus coined for this hypothetical substance (McCullagh, 1932). The hypothesis fell into disrepute and remained dormant until the 1970’s. The pertinent literature covering this has been authoritatively reviewed, to which the interested reader is referred (Setchell and Main, 1974; Main et al., 1979; Franchimont et al., 1979b). Although earlier studies connected inhibin with the regulation of FSH secretion primarily in the male, there is now convincing evidence to show its existence in the female.

An interrelationship between immune and reproductive systems has been postulated, and involves, among others, bidirectional effects between gonads and thymus. To this effect a rat thymus fraction of about 28000 mol wt has been reported to inhibit the effect of hCG on in vitro suspension of Leydig cells. We have investigated the antigonadotropin activity of thymus extracts on rat testis receptors. Acetonic powder obtained from thymus of 14 day-old rats was separated by molecular sieve chromatography. The effect of the collected fractions on the 125 I-hCG binding to receptor sites in rat testes was evaluated. A fraction corresponding to 27000–28000 mol wt named thymus factor (TF), was found to inhibit the binding activity of 125 I-hCG to its testicular receptor. The inhibitory effect of TF on hCG binding is dose related. By Scatchard analysis a competitive interaction at the receptor level between TF and hCG was demonstrated. The Ka values of hCG binding were diminished in the presence of TF while no significative changes were detected in the number of receptor sites. Present results strongly suggest a modulation function of TF at the testis receptor level.

The effects of hCG (CR119) and ethanol (20% in saline, v/v) on the levels of gonadotropin receptor in mature rat testes are studied. The results indicate that 75 I.U. of hCG administered intraperitoneally is capable of depleting testicular tissue of all its gonadotropin binding sites within 24 hrs. after administration, and 11–14 days are required for these binding sites to be fully restored. The disappearance and reappearance patterns of testicular gonadotropin binding sites following in vivo ethanol administration are very similar to those seen after gonadotropin administration, the only difference being that ethanol is much less immediate in its effect. The level of hLH in the serum of these rats is unaffected by the ethanol administered which seems to indicate that reduction of the number of gonadotropin binding sites in testes brought about by ethanol is through membrane-mediated mechanism. For rats receiving gonadotropin injections, no correlation sseems to exist between the concentration of gonadotropin in serum and the level of detectable gonadotropin binding sites in the testis.

The unexpected finding that LHRH (luteinizing hormone-releasing hormone) agonists block testicular testosterone secretion and induce a marked loss in ventral prostate weight in the adult rat (1–3) opened the possibility of a new approach in the treatment of androgen-dependent diseases, especially cancer of the prostate. Fortunately, among all species studied, man is the most sensitive to the inhibitory effect of LHRH agonist treatment on testicular androgen formation and medical castration can be easily achieved without any side effects other than those related to hypoandrogenicity (3–5).

It is well known that testicular function is controlled by hormones of the anterior pituitary gland. The absolute requirement for these hormones is demonstrated by the rapid atrophy of the sex accessory organs and inhibition of spermatogenesis following hypophysectomy (Wood and Simpson, 1961). The production of testicular steroids, in particular testosterone, is under the control of luteinizing hormone (LH), which is able to stimulate steroidogenesis in a variety of in vitro systems such as perfused testes, incubation of decapsulated testes, and suspensions of purified Leydig cells (Tsuhuhara et al,1977). There is good evidence showing that the action of LH in Leydig cells involves binding to specific receptors located on the plasma membrane, stimulation of cyclic AMP formation, and activation of protein kinase. The acute steroidogenic response of the testis to LH depends primarily on the activation of the enzymes controlling cholesterol side-chain cleavage, although there is also evidence for control mechanisms at a later step in the steroid biosynthetic pathway (Chasalow, 1979).

The interaction of follitropin (FSH, follicle-stimulating hormone) with testicular receptors has been studied most extensively in the rat model. Means and Vaitukaitis (1972) studied the binding of tritiated human FSH (hFSH) to receptors in immature rat testis. We have studied the interaction of biologically active, radioiodinated hFSH [125I]-hFSH with receptors in homogenates (Bhalla and Reichert, 1974a,b) and purified membranes from testes tubules of mature rats (Abou-Issa and Reichert, 1976). Desjardins et al. (1977) utilized radioiodinated human and rat FSH to study binding of the hormone to testis during maturation in the rat. It was found that the amount of labeled hormone bound to testis receptor increased slightly (P< 0.01) between 5 and 15 days and then remained relatively constant from 30 to 90 days. No apparent differences were noted in binding parameters between rat FSH and hFSH, although this was not rigorously examined. Considerable information is now available on the characteristics of the follitropin—receptor interaction in rat testes. A summary of reported values for K d and binding capacity is given in Table I. An outline of different conditions utilized to achieve maximum specific binding is also given in Table I. Binding of radiolabeled hFSH to rat testicular receptor is hormone- and tissue-specific and temperature- and pH-dependent, and apparently requires divalent cations for maximum binding, with no apparent specificity shown among Mg2+, Mn2+, and Ca2+ (Abou-Issa and Reichert, 1976).

Levels of FSH and LH were estimated in a crude powder of seminal plasma pooled from bulls. The FSH concentration in the seminal plasma was approximately 100 times greater than that in the circulation. The receptor-binding activity of LH in the seminal plasma was very low compared with that in blood. The FSH in bull seminal plasma was partially purified and found to be identical to ovine pituitary FSH in its ability to bind to bull testicular receptor, in its immunological reactivity and also in its chromatographic and electrophoretic properties. The partially purified LH in bull seminal plasma was more acidic than ovine pituitary LH as shown by disc electrophoresis. It showed low binding characteristics that were not parallel to pituitary LH in the specific radioreceptor assay. The significance of the inverse relationship between the levels of FSH and LH in seminal and blood plasma is unknown.

The concept that certain target tissues contain specific recep­tors for peptide hormones has been widely recognized (1). In almost all instances the cellular attachment of peptide hormones to the cell surface has been correlated with the activation of mem­brane bound adenylate cyclase and the production of 3′, 5′ cyclic adenosine monophosphate, the presumed intracellular mediator of hormone action (1). A similar sequence of events has been postulated to occur after testicular interstitial tissue was exposed to human chorionic gonadotropin (hCG) or lutenizing hormone (LH) (2, 3), or seminiferous epithelial cells were exposed to follicle stimulating hormone (FSH) (4). While these results support the notion that hCG/LH and FSH exert their effects on cells in the interstitial and tubular compartments of the testis, positive identification of the specific sites of high-affinity hCG/LH and FSH binding have not been defined at the tissue level in the same testis. Within this framework, it should be recognized that radioactive LH was localized selectively to Leydig cells (5–7) and that ferritin-labeled FSH appeared in the tubule wall and cytoplasm of Sertoli cells in rat testes (8). However, the latter findings may be ques­tioned in view of the possibility that metabolites of the labeled hor­mone were localized rather than the labeled hormone per se , since the hormone used was highly impure and massive amounts (i.e., 5 to 10 mg) were injected (8). Recent advances in the autoradio-graphic localization of peptide hormones in target tissues circum­vent the problems associated with hormone metabolites (9) and prompted us to reinvestigate the localization of gonadotrophic hor­mones in the testis.

Tissue receptor sites with high affinity for luteinizing hormone (LH) and human chorionic gonadotropin (hCG) have been defined in the interstitial cells of the testis, and also in the normal and gonadotropin-luteinized rat ovary (1–8). Comparable binding sites have also been demonstrated in the bovine corpus luteum, and in porcine granulosa cells during maturation in vivo and in vitro (9). The common receptor site for LH and hCG in these tissues is highly specific, with binding affinity only for molecules with the conformation characteristics of LH and chorionic gonadotropin. The biological specificity of hormone binding is demonstrable with LH from a wide variety of species, and with the placental gonadotropins of man, primates and the horse (2). The properties of the testis LH/hCG receptors, and their applications to radioligand-receptor assay, have been previously described in detail (2). In this chapter, the characteristics and application of gonadotropin receptors will be briefly reviewed, and more recent studies on the analysis of hormone-receptor interactions and the target cell responses to gonadotropins will be described.

Over the past several years this laboratory has been concerned with attempting to define in chemically precise terms the temporal sequence of events which occur upon the initial interaction of FSH with its testicular target cells. Previous studies have revealed that a single injection of this gonadotropin to immature male rats results in a rapid stimulation of RNA and protein synthesis (1–3). It is well established that FSH is a polypeptide hormone composed of two dissimilar subunits. Since studies with other peptide hormones such as insulin, glucagon, ACTH and LH have demonstrated that these hormones apparently do not enter the cell but instead bind specific receptors present on the surface of the target cell, we wished to investigate this possibility with respect to FSH and the testis. Our initial studies were made possible by the development of a procedure by van Lenten and Ashwell (4) for the radio-labeling of glycoproteins with tritium. Vaitukaitis and colleagues applied this procedure first to hCG (5) and then to FSH (6). Tritiation of the FSH involved oxidation of the carbohydrate side chain with periodate followed by reduction with tritiated borohydride. This procedure results in more than 85% of the tritium attached to the C7 position of sialic acid which comprises the terminal carbohydrate residue of the FSH side chain. FSH, radiolabeled in this fashion maintained a high biologic activity (1400 IU/mg) although the specific radioactivity was only about 0.25 mCi/mc.