Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus.

We recently reported isolation, characterization and synthesis of a novel ovine hypothalamic peptide with 38 residues which stimulates accumulation of cAMP in rat anterior pituitary cell cultures. The peptide was named PACAP38 (pituitary adenylate cyclase-activating polypeptide with 38 residues). The presence of another peptide corresponding to the N-terminal 1-27 residues (PACAP27) was also demonstrated. Both PACAP38 and PACAP27 have an amidated C-terminus. Antisera against synthetic PACAP27 were generated in rabbits. These antisera were tested for titer and specificity in enzyme-linked immunosorbent assay. One of the antisera (no. 88121-3) exhibited a high titer of antibody, which was specific to PACAP27 and PACAP38 with exception of slight cross-reactivity with ovine CRF (oCRF). Therefore, the antibodies against oCRF were removed from the antiserum using a solid phase method. Removal of oCRF antibodies was confirmed by enzyme-linked immunosorbent assay. A dense immunoreactive fiber network was found in both external and internal zones of the median eminence and pituitary stalk. The fibers were demonstrated to be in close contact with the hypophysial portal capillaries. The preabsorption of antiserum with vasoactive intestinal polypeptide or with the mixture containing TRH, LHRH, oCRF, ovine GH-releasing factor, somatostatin, and bovine thyroglobulin did not affect the immunostaining. On the other hand, the preabsorption of antiserum with an excess of PACAP27 or PACAP38 abolished the immunostaining. Therefore, the staining is considered specific for PACAP27 and PACAP38. Stained fibers were also present in the posterior pituitary. A dense fiber network was observed and the lateral hypothalamus the fibers appeared to cling to unstained neuronal cell bodies and their dendrites. In the lateral septum the fibers surrounded some blood vessels. Immunolabeled cell bodies were found in the paraventricular and supraoptic nuclei. These findings support the view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator.

A monoclonal antibody directed against CLIP (ACTH 18-39). Anatomical distribution of immunoreactivity in the rat brain and hypophysis with quantification of the hypothalamic cell group.

After the recent demonstration of the facilitatory effect exerted by corticotropin-like intermediate lobe peptide (CLIP or adrenocorticotropic hormone (ACTH) 18-39) on paradoxical sleep in the rat (Chastrette and Cespuglio, 1985), we undertook the production of monoclonal antibodies against this peptide. Wistar rats were immunized against CLIP and their spleen cells fused with mouse myeloma cells. After recloning, 25 supernatants were found to give positive immunohistochemical reactions in the rat brain. In immunohistochemical tests performed by preabsorption, the 25 supernatants presented similar properties, i.e. recognized CLIP, ACTH (1-39) and ACTH (25-39), but not ACTH (1-24) and the C-terminal fragment (34-39). We assume that the epitope(s) recognized by the 25 supernatants is (are) located between the amino-acids Asn25 and Ala34 of the CLIP molecule. The immunoreactivity observed in the rat brain and hypophysis with this antibody was distributed with a pattern quite similar to that described for anti-ACTH antibodies. A main group of immunoreactive cell bodies was located in the mediobasal hypothalamus and a small group in the nucleus of the solitary tract. Immunoreactive fibres were distributed from the olfactory nucleus to the spinal cord and formed particularly rich networks in the hypothalamus and preoptic area. Among other locations, immunoreactive axons were also present in the brainstem centres involved in the control of the sleep-waking cycle, which is in accordance with the influence of CLIP on paradoxical sleep. Using Abercrombie's formula, the number of immunoreactive cells in the mediobasal hypothalamus was estimated at about 3000 neurons. We conclude that our monoclonal anti-CLIP antibody can be considered as a good marker of proopiomelanocortin neurons.

Effect of dietary energy, body condition and calf removal on pituitary gonadotropins, gonadotropin-releasing hormone (GnRH) and hypothalamic opioids in beef cows.

Beef cows (n = 64) were slaughtered to evaluate effects of dietary energy and calf removal (CR) on hypothalamic and adenohypophysial endocrine characteristics. From d 190 of gestation until parturition, cows received maintenance (ME; n = 32) or low (LE; n = 32) energy diets (ME = 100%, LE = 70% NRC recommendations). After parturition, half (n = 16) of each prepartum diet group received low (LE; n = 32) or high (HE = 130% NRC; n = 32) energy diets. At 30 d postpartum, cows were slaughtered 0 or 48 hr after CR. Hypothalami [preoptic area (POA), hypothalamus (HYP), stalk-median eminence (SME)] and pituitaries were collected. Basal and K(+)-induced release of GnRH from SME, and pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) did not differ among groups (P greater than .05). Hypophyseal LH was correlated (P less than .01) with body condition score (BCS) at parturition and slaughter (r = .36 and .47, respectively). Prepartum LE diet increased (P less than .05) met-enkephalin in POA compared to prepartum ME (.59 +/- .05 vs. .44 +/- .04 pmol/mg) regardless of postpartum diet or suckling status. Concentrations of beta-endorphin in combined HYP + POA were decreased (P less than .05) by 48 hr CR (15.1 +/- 1.1 vs. 18.1 +/- 0.7 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat.

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 mu Ci/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-microns frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.

Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe.

A synthetic oligodeoxynucleotide sequence complementary to the mRNA for the adrenocorticotrophin (ACTH) precursor pro-opiomelanocortin (POMC) was end labelled using digoxigenin. The probe was used to detect POMC mRNA both on nitrocellulose filters and by non-isotopic in situ hybridisation (NISH) in tissue sections. Digoxigenin was identified using anti-digoxigenin alkaline phosphatase. The model system examined was the rat pituitary gland. Removal of both adrenal glands and dexamethasone administration were used to change the concentrations of POMC mRNA in the rat anterior lobe. The labelled probe reacted with a single band of appropriate molecular weight in Northern blot analysis. The distribution of signal in tissue sections and the changes induced by experimental manipulation were as predicted. The results indicate that this method of NISH will prove useful in the detection of specific messenger RNAs in tissue sections of buffered, formalin fixed, paraffin wax embedded material.

Luteinizing hormone in nutrient-restricted ovariectomized ewes.

This study was designed to evaluate profiles of serum concentrations of LH, pituitary content of LH and GnRH receptors, and hypothalamic content of GnRH in undernourished, ovariectomized ewes. In earlier studies, pulsatile secretion of LH diminished as duration of undernutrition progressed in prepubertal or adult ovariectomized ewes. Ewes having similar body condition scores (CS) of 5 to 9 (1 = extremely thin, 5 = moderate, 9 = obese) were fed maintenance or low-energy diets (100% and 60% of NRC requirements, respectively). Blood samples for analysis of LH were collected at 15-min intervals for 4 h at initiation of the project and immediately prior to slaughter. Serum concentrations of LH did not differ (P greater than .05) among groups at the initial sampling period. At slaughter, ewes with CS less than or equal to 2 (n = 7) had lost 26.8 kg (42% of initial weight). Ewes with CS greater than or equal to 3 (n = 12) had lost an average of 13.7 kg (18% of initial weight). Concentrations of LH in ewes with final CS greater than or equal to 3 was similar (P greater than .05) to that observed during the initial sampling period. However, release of LH was reduced (P less than .01) in ewes with CS less than or equal to 2 compared with ewes with CS greater than or equal to 3 (2.6 vs 9.5 and 3.2 vs 10.5 ng/ml for basal and mean concentrations, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic properties of Maharishi-4 against DMBA-induced mammary tumors in rats.

Maharishi-4 (M-4), an ayurvedic food supplement, was tested for anticarcinogenic and anticancer properties against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. The 6% M-4-supplemented diet protected DMBA-induced carcinogenesis by reducing both tumor incidence and multiplicity during initiation and promotion phases. The control animals who developed tumors when supplemented with M-4 diet for four weeks showed tumor regression in 60% of cases. There was no significant difference in the food intake or weight gain in rats who were on M-4-supplemented diet compared to control group. Possible mechanisms of action of M-4 are discussed.

Growth hormone (GH) autofeedback action in the neonatal rat: involvement of GH-releasing hormone and somatostatin.

It is known that in adult rats, GH by itself and by promoting secretion of the somatomedins acts at the level of the hypothalamus to trigger release of somatostatin and decrease output of GH-releasing hormone (GHRH), thereby inhibiting further secretion of GH. To assess whether these mechanisms are already operative in the early postnatal period, we have evaluated the effect of short-term administration of GH in 10-day-old rats. Twice-daily s.c. administration of 25 micrograms human GH/rat, from days 5 to 9 of life, significantly reduced pituitary content of GH, decreased hypothalamic levels of GHRH mRNA and abolished the in-vivo GH response to a challenge dose of GHRH (20 ng/100 g body weight, s.c.). GHRH (20 ng/100 g body weight, twice daily, s.c.) given concomitantly with the GH treatment, completely counteracted the inhibitory effect of the latter on pituitary content of GH and restored to normal the in-vivo GH response to the GHRH challenge. These data indicate that impaired secretion of GHRH is involved in the inhibitory effect elicited by GH treatment in infant rats. However, concomitant involvement of hypothalamic somatostatin as a result of GH treatment cannot be ruled out. In fact, pituitaries from rats pretreated with GH responded in the same manner as pituitaries from control rats to the GHRH challenge in vitro.

Complete purification of two identical Na(+)-pump inhibitors isolated from bovine hypothalamus and hypophysis.

We have completely purified, in parallel, a low molecular weight, non-specific, non-lipidic, Na+,K(+)-ATPase inhibitory factor from bovine hypothalamic and pituitary tissues. In the final purification step we obtain, from both tissues, a single, homogeneous peak, with a maximal absorbance at 247 nm. This factor, at physiological concentrations of potassium (5-25 mM), inhibits in a dose-response manner Na+,K(+)-ATPase and displaces ouabain from its receptor at the enzyme structure. The factor isolated from both tissues is identical, being the specific activity per weight of tissue higher in hypophysis. No factor was found in cerebral cortex, used as tissue control.

Reserpine-induced decrease in type I and II corticosteroid receptors in neuronal and lymphoid tissues of adrenalectomized rats.

The effect of the biogenic amine depleting drug, reserpine, on the concentration of type II corticosteroid receptors (i.e., glucocorticoid receptors) in neuronal (hippocampus, frontal cortex, hypothalamus), lymphoid (circulating lymphocytes, spleen, thymus) and pituitary tissues as well as hippocampal type I (i.e., mineralocorticoid) receptors was examined in adrenal-intact and adrenalectomized (ADX) rats. Reserpine (2 mg/kg) or vehicle was administered to adrenal-intact rats for 2 consecutive days. Following the second injection rats were ADX and sacrificed 24 h later. Reserpine significantly decreased type I and II hippocampal receptors as well as type II receptors in frontal cortex, hypothalamus, lymphocytes and spleen. Since the reserpine-induced decreases in receptor content could be due to reserpine-induced elevations in circulating corticosterone levels, reserpine (2 mg/kg) or vehicle was administered to 1-day ADX rats which were then sacrificed 2 days later (i.e., 3 days post ADX). A 1-day ADX control group was also included. The 3-day ADX regimen produced significant or nearly significant increases in type II receptors in hippocampus, frontal cortex, hypothalamus, lymphocytes and spleen in vehicle-treated rats. Reserpine attenuated the ADX-induced upregulation of type II receptors in hippocampus, frontal cortex, lymphocytes and spleen, but had no effect on the ADX-induced upregulation of type II receptors in the hypothalamus. The ADX-induced increase in hippocampal type I receptors was not affected by reserpine treatment. In a final experiment, reserpine (2 mg/kg) or vehicle was administered immediately after ADX and rats were sacrificed 24 h later in order to assess the effect of reserpine on basal (i.e., nonupregulated) corticosteroid receptor levels in the absence of circulating corticosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Galanin-like immunoreactivity is influenced by estrogen in peripubertal and adult rats.

Galanin gene expression in the anterior pituitary is potently stimulated by estrogen in adult rats. To evaluate the influence of estrogen on galanin during the peripubertal period 30- to 32-day-old female rats were treated with pregnant mare serum gonadotropin (PMSG, 10 IU s.c., 10.00 h). Galanin-like immunoreactivity (galanin-LI) in hypothalamic and pituitary tissues was evaluated 1, 2 or 3 days after PMSG treatment between 17.00 and 19.00 h. The PMSG treatment stimulated 17 beta-estradiol secretion, which induced a midafternoon LH surge 2 days after the PMSG treatment. Concentrations of galanin-LI at the time of this LH surge were elevated 82% in the anterior pituitary and 58% in the hypothalamus (without the median eminence) when compared to saline-treated female rats. On the 3rd day after the PMSG injection, galanin-LI was increased 236% in the anterior pituitary, 88% in the neurointermediate lobe and 39% in the median eminence compared to saline-treated female rats. These changes in galanin-LI were not observed in similarly aged male rats or ovariectomized rats treated with PMSG. In adult male rats, daily injections with 17 beta-estradiol valerate (10 micrograms/daily s.c.) for 1 week increased galanin-LI in the median eminence and neurointermediate lobe to an extent similar to that seen in juvenile female rats following PMSG treatment. In contrast, the high serum levels of 17 beta-estradiol achieved after 17 beta-estradiol valerate treatment increased galanin-LI in the anterior pituitary 65-fold. These studies indicate that galanin-LI is influenced by estrogen in peripubertal and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary.

GnRH-associated peptide (GAP)-like immunonreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14-69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.

Effects of vitamin A deficiency on the function of pituitary-gonadal system in male rats.

The effects of vitamin A deficiency on the pituitary-gonadal function were examined by measurements of serum and pituitary level of pituitary hormones and serum testosterone concentration, and by investigations of histological changes in the testis and the pituitary gland in vitamin A-deficient (VAD) and supplemented (VAS) rats. The growth of VAD rats was retarded and their body weights were decreased after 9 weeks of experiments and attained about one half of the weight of control animals at 12 weeks. In the VAD rats, serum testosterone concentrations were decreased significantly compared with those in the VAS controls. Serum and pituitary concentrations of GH were significantly lower but those of LH were slightly lower in the VAD rats than those in the controls, while the serum FSH concentration was significantly higher than that in the control rats. The seminiferous tubules in the testes of VAD rats were comprised largely of Sertoli cells and a reduced number of spermatogonia and contained fibrous formation in their lumen. In the pituitary gland, GH cells were significantly reduced in number in the VAD rats, but gonadotropic (GTH) cells were increased remarkably in size and number, showing hypertrophy and vacuolation similar to those in castration cells. The cytological changes in the pituitary gland and the increased discharge of FSH represent a secondary and compensatory change similar to that seen following castration and vitamin E deficiency.

[A study on PP family peptide receptor: I. The distribution of peptide YY (PYY) receptor in the brain].

Peptide YY (PYY) was discovered in the porcine gut by Tatemoto in 1982. PYY has recently been found in the central nervous system. This has provoked studies of PYY effects when centrally administrated. We investigated the specific binding of radioactive PYY (125I-PYY) to brain membranes in pigs and dogs. PYY chiefly bound to the hippocampus, as well as to the pituitary gland, hypothalamus, and amygdala, suggesting that PYY acts on the limbic-hypothalamic-pituitary axis. PYY binding in the brain had a high-affinity and a low-affinity component (dissociation constant, 1.39 X 10(-10) M and 3.72 X 10(-8) M, respectively). The binding sites were highly specific for PYY and neuropeptide Y (NPY), but not for pancreatic polypeptide (PP) or other peptide hormones such as cholecystokinin octapeptide, methionine enkephalin, adrenocorticotropic hormone, and thyrotropic releasing hormone. The similar affinities for PYY and NPY imply that these peptides regulate brain functions through interaction with common receptor site(s).

Gonadal steroids and anterior lobe dynorphin in the male rat.

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.

Non-isotopic RNA probes. Comparison between different labels and detection systems.

Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive 35S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.

Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium.

PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone-like immunoreactivity in brains of tetrapod vertebrates.

Heat-stable nondialyzable immunoreactive (IR) parathyroid hormone (PTH)-like activity, that coelutes with authentic PTH on Sep-Pak C18 columns and reverse-phase high-performance liquid chromatography, was measured in the brain, hypothalamus, and pituitary of amphibian, reptilian, avian, and mammalian species, using two specific antisera raised against the 48-64 region of the intact PTH molecule. In each case the IR PTH concentration was greater than that present in peripheral plasma and in rats was not affected by dietary calcium status. Extracts of muscle, liver, and kidney tissue were without IR PTH activity. These results demonstrate the extraparathyroidal occurrence of PTH-like peptides in nontumorous neuroendocrine tissues of vertebrate species. These findings may have evolutionary significance, since IR PTH was present in the brain and plasma of species that lack encapsulated parathyroid glands.

Changes in testicular and serum hormone concentrations in the male rat following treatment with m-dinitrobenzene.

m-Dinitrobenzene (m-DNB)-induced testicular atrophy has been attributed to a direct effect upon the germinal epithelium. However, such degenerative changes in the germinal epithelium should induce shifts in the testicular hormonal milieu, which would in turn alter the hypothalamic-pituitary gonadal axis in general. This study evaluated the endocrine status of male rats (killed 3 hr, 24 hr, 1 week, and 2 weeks) following a single oral dose of m-DNB (32 mg m-DNB/kg). Serum and pituitary leuteinizing hormone, follicle-stimulating hormone (FSH), and protactin and hypothalamic gonadotropin-releasing hormone (GnRH) concentrations were determined. Testosterone and androgen-binding protein concentrations in serum, interstitial fluid, seminiferous tubule fluid, and caput epididymis were also determined. In vitro basal and hCG-stimulated testosterone release was determined in the decapsulated testis. Results of the present study indicate that pituitary hormone concentrations and hypothalamic GnRH were unaffected after a single oral dose of m-DNB. Serum FSH was elevated at 2 weeks. There was a transient decrease in serum testosterone at 24 hr, which returned to control values at 1 and 2 weeks. Interstitial fluid, seminiferous tubule fluid, and caput epididymal testosterone concentrations were increased at 1 and 2 weeks. Basal testosterone release in vitro was increased at 2 weeks, while hCG-stimulated testosterone release was increased at 1 and 2 weeks. Androgen-binding protein concentrations in serum and interstitial fluid were increased at 1 and 2 weeks. Androgen-binding protein was increased at 24 hr and 1 week in seminiferous tubule fluid, but returned to control concentrations by 2 weeks. However, the total tubular content of androgen-binding protein was dramatically decreased at 2 weeks. Androgen-binding protein in the caput epididymis was unaltered following m-DNB treatment. These data demonstrate that m-DNB exerts a direct effect on the testes and not through alterations in hypothalamic and pituitary control of gonadal function.