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SUMMARY It is shown that anterior pituitary tissue grafted into hypophysectomized rats and lacking any connexion with the median eminence does not retain any somatotrophic or gonadotrophic activities, but has some adrenocorticotrophic (ACTH) and thyrotrophic (TSH) activities. This conclusion is based on: (1) the partial maintenance of thyroid and adrenal weight; (2) the results of studies on 131I uptake and on TSH concentration in the grafted pituitaries; (3) the persistence of significant adrenocorticotrophic responses to Pitressin, lysine-vasopressin and Guillemin's fraction D; (4) the presence of histological and histochemical signs of activity in the grafted gland. The subnormal levels of secretion observed in the pituitary grafted into sites far removed from the sella turcica, seem to depend on the lack of some neurohumoral stimulus which normally activates the intact pituitary via the hypophysio-portal vessels. Evidence is given that vasopressin may be the hypothalamic neurohumor involved in ACTH secretion, and that the ACTH-releasing activity of Guillemin's fraction D may be accounted for by its vasopressin content.

Pituitary autografts placed under the renal capsule of adult female rats in estrus were found to produce luteotrophin. Indirect evidence indicates that autografts in male and female rats which were operated on at puberty probably produce this hormone also. Studies on both the adult and “pubertal” animals indicate that pituitary autografts produce the other anterior lobe hormones either at a very low level or not at all.

The in vitro release of FSH from rat pituitary glands, when stimulated by a specific releasing factor, viz., follicle-stimulating hormone releasing factor (FRF), or nonspecific factors such as cyclic 3′,5′-AMP (cAMP), or high [K+] medium, required the presence of Ca++ in the external medium. Ca++ together with cAMP probably plays an active intracellular role in the processes leading to the release of FSH. No definite conclusion can be made as to the requirement for Mg++ in these processes. The synthesis (or activation) of FSH, observed during incubation in the presence of FRF, was also Ca++ dependent. However, further experiments are necessary in order to find out whether the presence of Ca++ is required directly for the synthesis of FSH, or whether the synthesis is only a secondary phenomenon arising from the release of FSH. In the latter case, the lack of Ca++, which inhibited the release of FSH, would consequently inhibit its synthesis. (Endocrinology 86: 761, 1970)

The histochemical distribution and quantitative chemical and histochemical changes in acid phosphatase activity were studied in rat pituitary and thyroid glands in various phases of secretory activity. The histochemically determined enzyme activity paralleled hormonal secretion. This could be confirmed chemically for the thyroid. The acid phosphatase activity appeared to be localized in the Golgi apparatus in these organs.

Rat pituitary, hypothalamic and cerebral cortical minces are demonstrated to effect the conversion of testosterone (T) to 5α-androstan-17β-o1-3-one (DHT) and Δ4-androstene-3,17-dione (Δ4A). The pituitary effected the greatest conversion of T to both DHT and Δ4A. The hypothalamus converted more T to DHT than did the cerebral cortex; but the conversion of T to Δ4A in these two tissues occured to approximately the same extent. Analysis of the time course of the conversion of T to DHT in these tissues demonstrated the conversion to increase in a linear fashion over 180 min., while the maximum conversions in the hypothalamus and cerebral cortex were reached by 120 min. The conversion of T to DHT and Δ4A was also demonstrated in the human fetal hypothalamus and pituitary, and in the distal femoral epiphysis of the growing rat. In the femoral epiphysis, 5α-androstane-3α,17β-diol was also identified.

The 00 LH double-antibody RIA system developed by Niswender at al. (1968a) has been tested to show its specificity for LH in the hamster. In this system hamster sera, hamster pituitary homogenates, sheep pituitary LH standard, and rat pituitary LH standard all show parallel dose-response curves. Hamster sera, TSH, and FSH do not appear to interfere with the assay of LII. Reproducibility and precision of the assay are within acceptable limits. The system reveals highly signfficant increases in serum LH after ovariectomy, during the course of proestrus and after in vivo administration of purified LH-releasing hormone. Blood samples were collected at different times up to 1 hr after a single iv, ip, or im injection of synthetic LH-RH. Only iv administration of the releasing hormone caused a rapid rise in circulating LH. At all times up to 1 hr the response seen after an im injection was contained within the dome of the curve representing the response observed after iv administration. Responses seen after ip and im injections were similar.

SUMMARY Anterior pituitaries from young female rats were incubated in Krebs-Ringer solution at 37° for 4·5–5 hr. A purified acetic acid extract of pig hypothalami was added, at various concentrations, to the incubation medium. The results show that the purified hypothalamic preparation increased the growth-hormone content in the supernatant of pituitary homogenates, suggesting an enhancement of hormone synthesis by the hypothalamic preparation. It is also suggested that the hypothalamic extract when incubated with pituitaries causes an increase in the rate of the release of growth hormone with prolonged incubation.

A sensitive and specific radioimmunoassay for rat thyrotropin (TSH) has been developed which utilizes 125I-mouse thyrotropic tumor TSH, antibovine TSH serum and USP bovine TSH as a standard. TSH in serum and pituitary homogenates was found in fractions corresponding to those previously shown to contain biological activity after gel filtration on Sephadex G-200. Serum TSH in normal rats after decapitation ranged from 15 to 66 μU/ml. During pentobarbital anesthesia, serum TSH was significantly lower (10–37 /μU/ml). Mean normal rat pituitary TSH content was 44 mU/gland. After 50μg thyroxine (T4) every other day for 14 days, serum TSH became undetectable (

SUMMARY Antiserum prepared by the immunization of rabbits, with homogenates of rat anterior pituitary gland according to a procedure previously outlined, was absorbed with homologous serum proteins and tissues to remove various non-specific antibodies. Varying levels of plasma from gonadectomized male rats, containing high levels of gonadotrophic hormone activity, were injected into hypophysectomized, immature female rats. The simultaneous injection of antiserum resulted in complete neutralization of gonadotrophic hormone as judged by the inhibition of ovarian and uterine weight responses and the extent of follicular development in the ovaries of the rats used for the bioassay. The degree of inhibition was dependent upon the relative amount of antiserum employed. Normal rabbit serum did not have any inhibitory effect. No detectable non-specific or toxic effects were noted in the animals injected with antiserum.

SummaryAnterior pituitary explants from female immature, mature and postpartum lactating rats were cultured in vitro on “199” or Trowell T-8 medium at 35 ± 1°C for periods of 6-14 days. Medium was assayed for prolactin by the intradermal pigeon crop method. Pituitary explants from mature female rats, gassed continuously with 95% O2 − 5% CO2 released 5-10 times more prolactin into medium each day than explants exposed only to air atmosphere. The amount of prolactin per AP recovered each day from the culture medium, under continuous gassing, was 2-3 times greater than that present in a fresh AP prior to culture. This proves that prolactin was actively synthesized and released and not merely “leached” out from AP explants. Explants from immature rats elaborated about half as much prolactin per AP per day as explants from mature rats, and ex-plants from lactating rats about twice as much as explants from mature rats. Assays of some explants at the end of culture revealed a low prolactin content, indicating ra...

THE present investigation was undertaken to ascertain the effects of cortisone treatment on the cellular composition of the rat adenohypophysis. In view of recent evidence for the influence of adrenal cortical steroids on the thyroid, the latter organ was also examined morphologically and its functional state was assessed by determining plasma and thyroid protein-bound iodine (PBI). MATERIALS AND METHODS One shipment of young adult male Sprague-Dawley rats was used in this study. In one experiment 12 rats received daily injections of 5 mg. cortisone acetate2 for 20 days. They were given ground Purina Laboratory Chow and tap water ad libitum. Since recent observations (Halmi, 1952 b) indicated that a rigid quantitative and qualitative control of the food intake of rats used in pituitary and thyroid studies is made necessary by the potential goitrogenicity of commercial regimens, each experimental animal was matched with an uninjected control which was pair-fed the same batch of diet.

SummaryCastration of adult male rats resulted in a 5-fold increase in pituitary LH concentration, but no change in pituitary FSH concentration. Estrone, testosterone, δ 4 Androstenedione and dehydroepiandrosterone inhibited the post-castration increase in pituitary LH and there was a relationship between dose and degree of inhibition. Those steroids which exhibited an androgenic effect increased the pituitary FSH concentration in the castrate pituitary, but a dose-response relationship was not discernible.

Melanocyte-stimulating hormone (MSH) activity in the pituitaries of albino rats did not change following the administration of morphine, decreased slightly with Nembutal, but fell markedly after the injection of both morphine and Nembutal. The additional use of ether did not alter pituitary MSH levels in rats which had received morphine. Treatment of rats with Nembutal, morphine and ether before administration of MSH-release inhibiting factor (MIF) resulted in a sensitive bioassay with little variability. (Endocrinology 83: 137, 1968)

Direct action of 6-dehydro-16-methylene-hydrocortisone (STC 407) and dexamethasone on the rat pituitary gland was investigated in vitro. CRF-induced ACTH release was suppressed after preincubation of the pituitary gland with 10 μg STC 407/ml or 1 μg dexamethasone/ml. These results suggest that both STC 407 and dexamethasone act directly on the pituitary gland.

The difficulty of using a single method for staining rat and human pituitary was investigated by testing separately and in different combinations the effects of the various components in the performic-acid-alcian-blue(P.F.A.B.)-periodic-acid-Schiff(P.A.S.) method (Adams and Swettenham, 1958). In this way the possibility of inhibiting or enhancing by one procedure the staining effects of the other could be assessed. The profound influence of fixation on these staining methods was demonstrated by employing the same set of staining methods on tissues fixed in different ways. The possibility that unbuffered performic acid might exert a considerable solvent action on certain proteins was considered to be related to the difficulty of applying the P.F.A.B.-P.A.S. method to the rat pituitary. Buffered performic acid has far less solvent action and the P.F.A.B.-P.A.S. method, modified by the replacement of buffered for unbuffered performic acid, proved to be successful in the demonstration of two types of basophil cells in both rat and man.

Malic dehydrogenase (MDH), lactic dehydrogenase (LDH), glucoses-phosphate dehydrogenase (G6PDH), β-galactosidase (β-GAL), β-glucuronidase (β-GLR) and β-glucosidase (β-GLU) in anterior, intermediate and posterior lobes of the male rat pituitary in normal, gonadectomized, thyroidectomized, or adrenalectomized animals were measured (10 days postoperatively in operated animals) by sensitive microchemical methods. In normal rats, anterior and intermediate lobe levels of MDH and G6PDH were significantly greater than posterior lobe, and anterior and intermediate lobe LDH were less than in the posterior lobe. Anterior and intermediate β-GAL and β-GLR were richer than posterior lobe, and for β-GLU the anterior was greater than the posterior lobe. Following gonadectomy, MDH rose in anterior and posterior lobe, decreased in intermediate lobe; β-GLR decreased in all lobes, β-GLU increased in all lobes, and β-GAL decreased in anterior lobe. After thyroidectomy, intermediate lobe levels of LDH, G6PDH, β-GAL, β-GLR and ...

SummaryPituitary tissue and rat pituitary extracts were incubated with dopamine in vitro. The results indicate that dopamine chemically inactivates ACTH and FSH when incubated in high concentration with these hormones for prolonged periods. The data fail to support a direct effect of dopamine on the pituitary release of ACTH and FSH, and dopamine does not alter the pituitary response to hypothalamic extract.

PURVES and Griesbach (1) have reported that in the rat pituitary two different types of gonadotrophic basophils could be found. One of these types was situated at the periphery of the gland in the proximity of the large portal vessels. The other type, less conspicuous in the normal animal, is located preferentially in the central regions of the anterior lobe. These two types of cells were named “peripheral” and “central” gonadotrophs and it was suggested that the peripheral type might be responsible for producing follicle stimulating hormone (FSH cell) while the central gonadotrophs might produce the luteinizing hormone (LH cell). Further evidence in support of this view has been obtained by Purves and Griesbach (2, 3). The existence of two types of gonadotrophs was also convincingly demonstrated by the electron microscopic studies of Farquhar and Rinehart (4).

Characteristics of the hydrolysis of histochemical substrates 5-bromoindoxyl acetate, naphthyl acetate, proprionate, butyrate, caprylate, laurate, myristate, and palmitate, acetyl and butyryl thiocholhie, chloroacetyl and trifluoroacetyl α-naphthylamide, benzoyl-arginine β-naphthylamide and proteinase substrates human hemoglobin and glycyl-phenylalanine amide by the rat pituitary tissue homogenate and DEAE-cellulose chromatography fractions were determined.

Hypothalamic extracts from the dog, rabbit, mouse, kitten, pigeon, and frog were found to decrease the growth hormone (GH) content of rat pituitary glands. Comparable cerebral cortical extracts were completely inactive in this test. These results broaden previous observations on the existence of a GH-releasing factor (GRF) in the hypothalami of mammals and show the presence of GRF activity in birds and amphibians.

SUMMARY Five monoamine oxidase inhibitors (MAOI) were tested for a correlation between MAO blocking potency, the effect on somatotrophic hormone (STH) production and metabolic effects on the pituitary. Mebanazine, N-acetyl-mebanazine and pargyline inhibited STH production, body weight gain and glucose-6-phosphate (G-6-P) dehydrogenase activity. MAOI which contain a hydrazine group (mebanazine and N-acetyl-mebanazine) also inhibited alkaline phosphatase activity as well as DNA and RNA formation in the pituitary, while pargyline, which contains an amino group, was inactive in these respects. Iproniazid and norpargyline were inactive with regard to STH production, body-weight gain and metabolic activities of the pituitary: alkaline phosphatase activity, G-6-P dehydrogenase activity, and DNA and RNA content. This suggests a relation between the ability of an MAOI to inhibit STH production and inhibition of the phosphogluconate oxidative pathway.

SummaryA specific rabbit anti-sheep LH serum (A/S) was implanted into the rat hypothalamic median eminence (ME) to investigate the mode of action of a “short” feedback of endogenous LH by neutraliz...

Cyclic rats known to release LH between 2:00 P.M. and 4:00 P.M. on the day of procstrus were tested for simultaneous ACTH release by measurement of adrenal ascorbic acid concentration between 3:30 and 4:30 P.M. NO depletion of ascorbic acid occurred, indicating that ACTH is not released simultaneously with LH. Pentobarbital treatment at 2:00 P.M. did not cause significant adrenal ascorbic acid depletion in rats of different cycle stages. Therefore, the ability of pentobarbital at 2:00 P.M. to block LH release in proestrous rats is not the result of a non-specific “stressing” action as has been suggested in the literature.

In an attempt to localize the corticotrophs of the rat pituitary, partially purified ovine CRF and five control materials (acid saline, Sephadex column eluate outside the CRF and vasopressin zones, 10M ammonium acetate in acid saline, sublimated 10M ammonium acetate, sublimated column eluate) were injected into the pars distalis, the pars intermedia, or the junction between the pars intermedia and the pars distalis. Acid saline was inert in all regions. The materials containing ammonium acetate stimulated at least some ACTH release. Following sublimation to remove ammonium ions, only CRF still stimulated ACTH release, and it was more effective at the pars intermedia or the junction of the pars intermedia and the pars distalis than it was in the body of the pars distalis. From these studies we have drawn two conclusions: 1) the pars intermedia or the nearby pars distalis region of the pituitary is more responsive to exogenous, hypothalamic CRF than is the bulk of the pars distalis; 2) it is necessary to re...

By means of fluorescent antiprolactin globulin and with appropriate controls, lactogenic hormone has been localized in granules and mitochondnia of acidophils of normal rat pituitary and in a transplantable rat pituitary tumor. Cells containing prolactin have been identified as acidophils by their reaction with orange C and by positive staining of their cytoplasmic granules with acid fuchsin in the Russell technique. Immunochemical data is presented reaffirming the heterogenous character of purified ovine prolactin. Evidence that this is associated with polymerization of prolactin is discussed.

Growth hormone-producing rat pituitary cells have been hybridized spontaneously with thymidine kinase deficient mouse fibroblasts. All clones isolated were made up of cells containing about 180 chromosomes and showing a combination of 2S mouse: 1S rat genomes. Ten percent of the chromosomes were lost by the 20th passage and about 40% were lost by the 45th passage with a preferential loss, during later passages, of rat chromosomes. Electrophoretic studies of adenosine deaminase and lactate dehydrogenase gave evidence that both rat and mouse genomes were functional; however, none of the hybrid clones, even after only 2 to 5 passages, produced growth hormone.

Pituitary homotransplants under the median eminence were capable of inducing a normal adrenal secretion of corticosterone in response to traumatic stress in hypophysectomized rats. Misplacement of the transplants by as little as 1–2 mm or damage to the hypophysial stalk of the host greatly reduced the ability of otherwise viable and healthy transplants to stimulate adrenal secretion. (Endocrinology 74: 939, 1964)

The GH 3 strain of rat pituitary tumor cells has been adapted to growth in suspension culture. The cells have been grown in this fashion for a year and have maintained a doubling time of 42–48 h. They have continued to produce both growth hormone and prolactin. When the cells were exposed to hydrocortisone (3 × 10 −6 M), GH production was stimulated by about 7-fold, and prolactin production was decreased by about 3- to 5-fold, while the cell doubling time was unaffected.

IN PREVIOUS REPORTS (1–5), there was described a condition resembling hypophysectomy which was produced by underfeeding rats. The atrophy of the endocrine organs of the rat resulting from chronic inanition has been designated pseudohypophysectomy not only because of its resemblance to the effects of hypophysectomy but also because it is believed that many of the effects of inanition are due to malnutrition of the hypophysis, resulting in a diminished secretion of hormones. The endocrine tissue changes are qualitatively similar in the two conditions but they are seldom so marked during chronic inanition as they are after hypophysectomy. Changes in other tissues such as the spleen, thymus,and liver are equally marked in both conditions and appear to be related to the loss in body weight. The purpose of the present study is to determine the effects of complete hormonal replacement therapy in the form of rat pituitary implants, in modifying the effects of complete starvation and of chronic inanition.

An investigation has been made of the long-term effect of low radiationr doses to the rat pituitary using a high energy deuteron beam. The pituitarics were irradiated when the rats were 28 days old. The animals were maintained up to 27 months thereafter. A total of 138 controls and 169 treated rats were studied. Groups varying in size from 7 to 20 rats at each dose of radiation were sacrified at intervals of about 3 months. The endocrine changes became increasingly more prominent with increasing dosage and with time. Within certain dose ranges the same terminal results were obtained, though the lower the close the longer theperiod which elapsed before the maximum effect resulted. The effect of the radiation on pituitary structure was as follows:decrease in size of all lobes; loss of demarcation between lobes; increased connective tissue in surrounding membranes and supporting framework and frequent small hemorrhages. In the anterior lobe the change effected in the shortest time and at the lowest dose was ...

SUMMARY Thyroidal uptake and release of 131I were used to study the effects of prolonged infusion of porcine hypothalamic extract on the ability of rat pituitary autografts to secrete thyrotrophin (TSH). The extract used contained insufficient TSH to induce release of thyroidal 131I in hypophysectomized rats. After infusion for 10 days, the time of maximal uptake of 131I and onset of 131I release was significantly shortened by infusion of hypothalamic, but not cerebral cortical extract, when compared with non-infused autografted controls. The rate of release of thyroidal 131I was significantly increased by the infusion of hypothalamic extract so that by 96–120 h after the administration of 131I the rate of release was not significantly different from that in intact controls. Accelerated thyroidal release of 131I began 42–48 h after the application of hypothalamic extracts to pituitary autografts and fell rapidly after withdrawal of the extract. At the end of 14–17 days of infusion sections of the autografts contained aldehyde-fuchsin positive staining basophils.

Secretory granules have been seen within components of the Golgi bodies of rat pituitary acidophils and mouse pancreatic acinar cells. The fact that secretory granules are much more frequently encountered within Golgi components under conditions of increased secretory activity suggests that granule formation may occur within the Golgi apparatus in these two types of cells.

Luteinizing hormone preparations from a variety of sources were compared with respect to the slopes of their radioligand uptake inhibition curves. The system utilized human luteinizing hormone as the radioligand and homogenates of testis from mature rats as the receptor source. Based on the weighted mean values from replicate slope measurements, four major groups could be detected. In order of decreasing slope steepness, these were: Group I = human urinary LH, monkey pituitary LH, human pituitary LH and human chorionic gonadotropin; Group II = bovine pituitary LH, ovine pituitary LH, rat pituitary LH and equine pituitary LH; Group III = rabbit pituitary LH and chicken pituitary LH; Group IV = porcine pituitary LH and canine pituitary LH. There was no overlap of 95% fiducial limits for calculated slope values between groups. These groupings are at variance in some instances with those of a previous report (Endocrinology 73: 509, 1963), based on slopes of dose response curves obtained using the ventral pros...

IN STUDIES of changes in the ACTH content of rat pituitary glands, the resting value and its usual range must be firmly established. There are anly a few reports of such values. For example, Burns, Merkin, Sayers, ond Sayers (1) found about 100 mu. of ACTH per rat pituitary, and Sydnor and Sayers (2) reported a value of 70 mu. per gland. More recently, Saffran and Schally (3), using a different method of assay, found values ranging about 300–400 mu. per 5–6 mg. rat gland. The discrepancy could result from different methods of extraction and/or different bioassay procedures. In the course of experiments designed to detect the source of the disagreement, we found that the extraction procedure is probably the major variable and that it is possible to extract more than 1000 mu. from each rat pituitary gland—10 times as much as the values first reported by Burns et at.!

Rat pituitary was cultured for 4, 6 or 9 days at 37 C, and rat hypothalamic or cerebral cortical extract was added to the medium after 1, 3 or 6 days of culture. TSH was assayed by the McKenzie method. Release of TSH into the medium by pituitary exposed to rat cerebral cortical extract (Control) fell to very low levels after the first 3 days of culture. When rat hypothalamic extract was incorporated into the medium at the end of 3 or 6 days of culture, output of TSH by the pituitary for the following 3 days increased to levels equal to or exceeding those of the first 3 days. The total amount of TSH removed from the medium and pituitary explants cultured with hypothalamic extract for 4 or 6 days was 50–97 % greater than found initially in fresh rat pituitary. These results indicate that a rat hypothalamic extract can increase synthesis as well as release of TSH by the rat pituitary in vitro. (Endocrinology 78: 1002, 1966)

The aim of the study was to attempt the simultaneous translation in the reticulocyte lysate of GH and PRL mRNA from cultured GH3 cells and from tumors grown in the rat by transplantation with the same cell line. The results show that, while both mRNA activities are present in the cultured cells, only GH mRNA is detected in the transplantable tumor.

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.

Acid extracts of rat pituitary neuro-intermediate lobes have been shown by ion-exchange chromatography and radio-immunoassay to contain predominantly the inactive derivatives of beta-endorphin, alpha, N-acetyl beta-endorphin 1-27 and alpha, N-acetyl beta-endorphin 1-26; the biologically active form, beta-endorphin 1-31, is a minor component. In contrast, it was found that beta-endorphin generated in neuro-intermediate lobe cells in monolayer culture was less processed: the principal peptides related to bioactive beta-endorphin 1-31. When the cultured cells were incubated in the presence of 10(-5) M dopamine or 10(-6) M alpha-ergocryptine there was a marked increase in the degree of proteolysis and acetylation: the processing pattern reverted to that characteristic of the neuro-intermediate lobe in situ, with alpha-N-acetyl beta-endorphin 1-26 and alpha, N-acetyl beta-endorphin 1-27 as the prominent peptides. The results demonstrate that dopaminergic agents can influence the processing of beta-endorphin-related peptides in rat pars intermedia, indicating a new level at which the bioactivity may be regulated.

To further explore the mechanism of steroid feedback in male, the effects of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the rates of alpha- and lutropin (LH)beta-chain synthesis, neosynthesized subunits and radioimmunoassayable LH release into the medium were studied in the cultures of anterior pituitary cells from orchiectomized and intact rats. Polypeptides were [35S]methionine-labeled, immunoprecipitated separately in the medium and cells, then after SDS-PAGE precisely quantified. The total (medium + cells) radioactivity incorporated in the absence of GnRH into alpha- and LH beta-subunit was increased in orchiectomized rat cells vs. intact rat cells. GnRH stimulated the synthesis of both subunits, whether cells were from normal or castrated rat. T suppressed basal and GnRH-enhanced synthesis of both subunits in castrated rat cells. The values became closed to those observed in the normal rat cells. Also release of neosynthesized subunits from castrated rat cells into the culture medium was inhibited by T. In contrast, T did not change the basal and GnRH-induced radioimmunoassayed LH release. These results show that T can inhibit directly, at the pituitary level, alpha- and LH beta-subunit synthesis and neosynthesized but not stored LH release. They could explain, at least in part, no correlation between modifications of GnRH and LH secretion observed in vivo in response to T replacement.