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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).

Nuclear receptor 4A1(NR4A1) (testicular receptor 3 (TR3), nuclear hormone receptor (Nur)77) is a member of the nuclear receptor superfamily of transcription factors and is highly expressed in multiple tumor types. RNA interference studies indicate that NR4A1 exhibits growth-promoting, angiogenic and prosurvival activity in most cancers.Studies on several apoptosis-inducing agents that activate nuclear export of NR4A1, which subsequently forms a mitochondrial NR4A1-bcl-2 complex that induces the intrinsic pathway for apoptosis are discussed. Cytosporone B and related compounds that induce NR4A1-dependent apoptosis in cancer cells through both modulation of nuclear NR4A1 and nuclear export are discussed. A relatively new class of diindolylmethane analogs (C-DIMs) including 1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) (NR4A1 activator) and 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) (NR4A1 deactivator) are discussed in more detail. These anticancer drugs (C-DIMs) act strictly through nuclear NR4A1 and induce apoptosis in cancer cells and tumors.It is clear that NR4A1 plays an important pro-oncogenic role in cancer cells and tumors, and there is increasing evidence that this receptor can be targeted by anticancer drugs that induce cell death via NR4A1-dependent and -independent pathways. Since many of these compounds exhibit relatively low toxicity, they represent an important class of mechanism-based anticancer drugs with excellent potential for clinical applications.

Cloned cDNA encoding the rat Sertoli cell receptor for FSH was isolated from a cognate library and functionally expressed in cultured mammalian cells. The FSH receptor (FSH-R), as predicted from the cDNA, is a single 75K polypeptide with a 348 residue extracellular domain which contains three N-linked glycosylation sites. This domain is connected to a structure containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. Thus, the FSH-R is identical in its structural design to the LH/CG receptor (LH/CG-R). Furthermore, both receptors display 50% sequence similarity in their large extracellular domains and 80% identity across the seven transmembrane segments. Expression of the cloned cDNA in mammalian cells conferred FSH-dependent cAMP accumulation. The selectivity for FSH is attested by the fact that the related human glycoprotein hormones human CG and human TSH do not stimulate adenylyl cyclase in FSH-R expressing cells even when these hormones are present at high concentrations.

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).

Calcium-dependent transglutaminase (TGase) activity, determined by incorporation of [1,4-14C]di-aminobutane dihydrochloride (putrescine) into casein, was demonstrated in a light membrane fraction prepared from bovine calf testicular homogenates. Purification of these membranes by sucrose density gradient centrifugation produced a follicle-stimulating hormone (FSH) receptor-enriched fraction containing TGase activity which cosolubilized with the FSH receptor and could be incorporated with detergent-solubilized receptor into liposomes. In the present study, we show that calcium increases specific binding of FSH to receptor in a concentration-related manner, and is associated with an increase (13.2-fold at 20 mM) in the affinity (Ka) of the receptor with no significant (P>0.05) change in receptor concentration. Treatment of the light membrane fraction with monodansylcadaverine (MDC, 1 mM), a specific inhibitor of TGase, did not affect specific binding of FSH, but resulted in only a 3.9-fold increase in Ka at 20 mM calcium with no change in receptor concentration. Specific binding of FSH to receptor at 4°C was also enhanced by calcium. Scatchard analysis of competitive binding inhibition data showed a Ka at 20 mM calcium similar to that observed with MDC. Dissociation of [125I]hFSH-receptor complexes formed at 30°C in the presence of calcium was significantly less than dissociation of complexes formed at 30°C in the absence of calcium. When [125I]hFSH-receptor complexes were formed at 30°C in the presence of calcium and dissociated in calcium-deficient buffer, dissociation increased 3-fold. Similar results were obtained in the presence of MDC. The ability of both calcium and the TGase inhibitor MDC to modulate FSH binding and dissociation suggests that calcium- and temperature-dependent TGase activity may explain the progressively irreversible binding of FSH to its receptor observed in earlier studies conducted at 30°C or above. Our data support a model whereby calcium may stabilize binding of FSH to its testicular receptor via activation of TGase-catalysed isopeptide bond formation.

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.

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.

Defective luteinizing-hormone-mediated cAMP generation in rat testis was previously demonstrated by us in acute and chronic uremia. This defect was abolished by administration of 1,25-dihydroxycholecalciferol (1,25-dihydroxycalciol). In the present study, we furnish evidence for a cytosolic 1,25-dihydroxycalciol receptor in the rat testis. Testes of non-rachitic, rachitic or acutely uremic male Sprague-Dawley rats were homogenized in 0.3 M KCl, 10 mM Tris/HCl, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4. Fresh purified cytosol was incubated with 1,25-[3H]-dihydroxycalciol (with or without 200-fold molar excess of unlabeled 1,25-dihydroxycalciol, subjected to 5–20% sucrose density-gradient separation (centrifugation with 255000xg, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule in testicular cytosol with the properties of the intestinal 1,25-dihydroxycalciol receptor. The binding macromolecule migrated at 3.5 S and could clearly be distinguished from the 6-S tissue-binding site for 25-hydroxycholecalciferol. The 1,25-dihydroxycalciol receptor was present in non-rachitic, rachitic and acutely uremic rats. Scatchard analysis of the receptor demonstrates high affinity and selectivity (1,25-dihydroxycalciol ≫ 25-hydroxycholecalciferol > 1 α-hydroxycholecalciferol > 24(R),25-dihydroxycholecalciferol), low capacity (Nmax= 82 fmol/mg protein) and an equilibrium constant of Kd= 2.6 × 10−10 M. The receptor is a protein according to enzyme degradation studies (trypsin, pronase, RNAase, DNAase). Competition studies with and without the alkylating reagent N-ethylmaleimide demonstrate a cysteine residue in or close to the binding site for 1,25-dihydroxycalciol. Binding of 1,25-dihydroxycalciol protects the receptor against inactivation with N-ethylmaleimide.

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...

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.

Adult rat testis contains a specific, high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) with properties similar to 1,25-(OH)2D3 receptors in other tissues. The receptor sediments at 3.5 +/- 0.2 S20,w in high-salt sucrose density gradients, but aggregates in low-salt gradients. Binding of 1,25-(OH)2D3 was abolished by trypsin, but not by DNase or RNase. Binding was also heavily reduced by the sulfhydryl alkylating agent, N-ethylmaleimide, and by the mercurial reagent, mersalyl, showing that free, reduced SH-groups are necessary for hormone-binding activity. The receptor shows high affinity for 1,25-(OH)2D3 (Kd = 3 X 10(-11) M), but low capacity (Nmax = 8 fmol/mg protein) and is specific for 1,25-(OH)2D3 (Affinity: 1,25-(OH)2D3 greater than 1,24(R),25-(OH)3D3 greater than 25-OH-D3 greater than 1 alpha-OH-D3 greater than 24(R),25-(OH)2D3 much greater than 17 beta-estradiol, testosterone, dexamethasone, R5020, progesterone). With 0.6 nM [3H]1,25-(OH)2D3 and at 0 degrees C, maximum specific binding was achieved after 4 h, and the occupied receptors were stable for more than 24 h. The dissociation of hormone-receptor complexes was temperature-dependent and very slow at low temperature (t1/2 (0 degrees C) much greater than 48 h). At 0 degrees C, the second order association rate constant and the pseudo-first order dissociation rate constant were 2.7 X 10(7) M-1 min-1 and 2 X 10(-5) min-1, respectively. Receptors for 1,25-(OH)2D3 are present in similar amounts in isolated seminiferous tubules and interstitial tissue of adult rats. No specific binding of [3H]1,25-(OH)2D3 could be detected in cultured immature Sertoli cells, cultured immature peritubular (myoid) cells or crude germ cells.

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.

Considerable data have accumulated over the last decade and a half to document that alcohol abuse is associated with and is the most probable cause of the Leydig cell failure that occurs in chronic alcoholic men.1,2 The mechanisms responsible for this adverse consequence of ethyl alcohol abuse on Leydig cell function extend to all cellular sites within the Leydig cell that are responsible for testosterone biosynthesis and also involve sites before (the pituitary and hypothalamus) and after (e.g., the liver, fat cells, skin) the Leydig cell that contribute to the androgen economy of the individual. This chapter reviews the considerable literature pertaining to this problem and presents the findings available concerning the adverse effects of ethanol abuse on ovarian function.

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.

The discovery of potent hypothalamic analogues led to extensive laboratory investigations, confirming that both agonistic and antagonistic analogues of luteinizing hormone-releasing hormone (LHRH) had inhibitory effects on rat prostate tumors [1–3]. Excitement generated by this significant finding stimulated implementation of a series of clinical studies in human prostate tumors designed to define the appropriate dose of LHRH analogues, establish a toxicity profile, and evaluate therapeutic activity. With an acceptable toxicity pattern and demonstrable clinical activity, LHRH analogues then entered clinical trials comparing their activity to the established therapeutic interventions, including orchiectomy or oral estrogens, for advanced prostate cancers to evaluate treatment morbidity, tumor responsiveness, time to disease progression, and the ultimate parameter of survival. In this chapter, the data generated by the LHRH clinical investigations in the treatment of prostate cancer will be summarized.