Prolactin (PRL) is one of the most versatile hormones of the pituitary in terms of biologic actions, but how a single molecule is able to evoke so many different responses in the organism is not known. Research in recent years has uncovered a surprising degree of structural polymorphism for PRL, the different forms having variable biopotencies. Such findings lend credence to the hypothesis that the molecular heterogeneity of PRL is one of the mechanisms for creating diversity in the biologic actions of this hormone.

A novel combination of two single cell assays allowed the simultaneous measurement of intracellular calcium concentration and hormone secretion in normal pituitary cells. [Ca2+]i was recorded using the fluorescent Ca2+ indicator fura-2 and digital imaging microscopy. This technique was combined with a reverse hemolytic plaque assay for growth hormone in order to identify somatotropes and quantitate the amount of hormone released. A dynamic profile of rhythmic calcium oscillations was found in spontaneously secreting somatotropes. Each somatotrope displayed a distinct frequency (one pulse every 5-30 s) and amplitude (range 50-450 nM) generated asynchronously from cell to cell. The amount of growth hormone (GH) released correlated directly with both the frequency and amplitude of calcium oscillations at the level of single GH cells. Furthermore, calcium excursions in somatotropes were rapidly suppressed by either (i) removal of extracellular calcium, (ii) somatostatin (1 mM), or (iii) the calcium channel blockers cobalt (2 mM) and verapamil (100 microM). These observations demonstrate that spontaneous calcium oscillations are characteristic for normal somatotropes. These oscillations are related to spontaneous hormone secretion and due to influx through calcium channels in the membrane. Somatostatin, the physiologic inhibitor of GH secretion, suppresses calcium transients. These findings suggest that the intracellular signaling information may be encoded both in the frequency and amplitude of calcium oscillations.

The mechanism by which gonadal steroids modulate GH secretion is not known. We have used the reverse hemolytic plaque assay to examine whether gonadal steroid-induced modulation of GH secretion is effected by changes in the population of somatotrophs and/or alterations in their secretory properties. Two groups of Sprague-Dawley rats were studied: group 1 (n = 6) comprised male (M), castrate (Cx), and testosterone-replaced castrate male (Cx + T) rats and group 2 (n = 5) consisted of male (M), female (F), and 17 beta-estradiol-replaced castrate male (Cx + E) rats. The number of plaque-forming cells (expressed as both absolute number and a percentage of all cells) was determined, and secretory status was assessed by measuring plaque areas in response to 0, 0.01, 0.1, 1, 10, and 100 nM GHRH. While mean basal GH plaque areas were similar among the treatment groups of group 1, the maximal GH plaque area was significantly decreased in Cx [16.8 +/- 2.4 vs. 26.4 +/- 3.9 X 10(6) microns2 (mean +/- SEM); P less than 0.05], but not in Cx + T (27.5 +/- 4.1 microns2) rats. The GHRH EC50 was unaffected by castration or T replacement. The percentage and absolute population of somatotrophs were reduced in Cx, but not in Cx + T, rats, while the numbers of lactotrophs remained unchanged in these treatment groups. For group 2, the mean peak GH plaque area was reduced in Cx + E (16.5 +/- 2.9 microns2; P less than 0.001) compared to that in M rats (36.2 +/- 2.3 microns2), but was not significantly different from that in F (13.0 +/- 1.5 microns2) rats. The EC50 was significantly (P less than 0.025) greater in Cx + E (10.9 +/- 2.3 nM) and F (7.9 +/- 1.6 nM) compared to M rats (2.8 +/- 0.7 nM). The absolute somatotroph and lactotroph populations were increased in Cx + E compared to M and F rats, as were the populations of other pituitary cell types. Testosterone enhances GH secretion by increasing the secretory capacity, but not the sensitivity, of somatotrophs to GHRH and by recruiting the function of a subpopulation of somatotrophs. Estradiol reduces the secretory capacity and sensitivity of somatotrophs to GHRH, but increases the population of somatotrophs, lactotrophs, and non-GH- and non-PRL-secreting cells.(ABSTRACT TRUNCATED AT 400 WORDS)

It is not known whether enhanced growth hormone (GH)-releasing factor (GRF)-stimulated GH release observed in the male reflects differences in somatotrope numbers and/or secretory response to GRF. We addressed this question by using the hemolytic plaque assay which allows quantification of hormone secretion by single pituitary cells. Time-course studies and GRF-GH concentration-response relationships (0.01, 0.1, 1, 10, 100, 1,000 nM GRF) in age-matched male and diestrous day 2 female rats were compared by quantitating the percent of GH plaque-forming cells, and measuring the plaque areas. The male pituitary contained a greater percent (P less than 0.05) of somatotropes (% of plaque-forming cells 45 +/- 2 vs. 27 +/- 4% in the female; mean +/- SE). GRF induced a greater concentration-dependent increase in plaque areas in the male. Maximal responses were attained at 10 nM GRF in both sexes. However, mean maximal plaque area was significantly greater (P less than 0.001) and the EC50 was significantly lower (P less than 0.05) in the male (0.25 +/- 0.09 vs. 1.78 +/- 0.64 nM in the female). The data suggest that the greater percent of somatotropes in the male and greater secretory capacity and sensitivity to GRF may contribute to sex-related differences in GH secretion in the rat.

Weanling male rats were trained to consume a control diet in one 4-hr meal a day. Rats were then fed one of three experimental diets for 7 days: (a) control diet minus B6 (-B6); (b) control diet (B6); or (c) control diet restricted to food intake of -B6 (B6R). Xanthurenic acid excretion was greater before and after a tryptophan load in -B6 than in B6R. Body weight gain, food intake, and food efficiency were not different between -B6 and B6. However, in B6R body weight gain, food intake and food efficiency were lower than that in both -B6 and B6. Serum glucose (12-16 hr after meal) and percentage body fat were similar in all three groups. No differences were found in organ size between the -B6 and both control groups indicating that short-term deprivation and deoxypyridoxine did not affect organ size. No significant differences were observed for serum growth hormone (GH), pituitary GH, serum prolactin (PRL), or pituitary PRL among the three groups. When meal fed, differences were minimized between B6 deprived and unrestricted control (B6) rats in body weight gain, food intake, food efficiency, and body composition.

A glycosylated form of human PRL (G-hPRL) was isolated from pituitary glands. The glycoprotein was separated from the major form of PRL on columns of lentil lectin-Sepharose 4B. The major form of PRL did not bind to the lentil lectin, whereas the glycosylated modification did and could be eluted with methyl-alpha-D-mannopyranoside. By gel electrophoresis in sodium dodecyl sulfate, a mol wt of 25,000 was estimated for the glycosylated PRL. The mol wt of hPRL is 23,000. In a RIA for hPRL, the glycosylated hormone was about one third as reactive as the principal form. Since there is only one Asn-X-Ser(Thr) sequence in hPRL, the asparagine at position 31 is the likely point of N-linked glycosylation.

The hemolytic plaque assay technique can be used to detect specific hormone release from single pituitary cells. Using antisera raised against murine GH or rat PRL, we have enumerated the active lactotropes and somatotropes from male and female rat pituitary glands. These studies reveal sex-related differences in the number of cells exporting GH and PRL among anterior pituitary cells in culture. In the presence of human GH-releasing factor (hGRF), the mean percentage of GH cells was 53% in males and 30% in females (P less than 0.005). The mean percentage of PRL cells was 15% in males and 39% in females (P less than 0.008). These values were not significantly altered when hGRF was omitted. The sum of GH and PRL cells identified in separate plaque assays significantly exceeds the number obtained when GH and PRL cells were determined concurrently with a simultaneous plaque assay for both hormones. This difference is dependent on the presence of hGRF, since there was no difference when hGRF was omitted. These data identify the mammosomatotrope in numbers lower than previous reports. By this approach, the mammosomatotrope subpopulation numbers about 5% of all cells in culture. In summary, we demonstrate a sex-related difference in the number of cells exporting GH or PRL among pituitary cells in culture. This difference corresponds with and may underly sex-related differences in the responsiveness of GH and PRL secretion from the pituitary gland. Furthermore, a minor subpopulation of normal pituitary cells appears capable of simultaneous secretion of both GH and PRL.

A form of rat PRL with a clip in its large disulfide loop, the so-called cleaved PRL, has been reported to have a greater mammogenic activity than the intact molecule. This study was undertaken to investigate the presence of cleaved PRL in the mouse pituitary gland with the purpose of correlating its concentrations with the incidence of mammary tumors. We could identify this molecule in the mouse pituitary by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although its concentration was not high enough for ready detection in crude extracts of pituitary tissues. Cleaved mouse PRL and its 16 K and 8 K fragments cross-reacted with antibodies to the intact molecule and, thus, cannot be differentiated from the latter by RIA. When pituitaries were incubated for 6 h with 14C-labeled amino acids, cleaved PRL from the pituitaries of male mice incorporated more radioactive amino acids than the corresponding molecules from female mice. However, treatments such as ovariectomy, ovariectomy plus estradiol benzoate, perphenazine, and 2-Br-alpha-ergocryptine affected the concentration of labeled cleaved PRL in the same manner as they did that of the intact molecule. The ratio of labeled cleaved PRL to the labeled intact molecule in mice with a high incidence of mammary tumors (C3H/St) was not much different from that in mice with a low incidence (C57BL/6J) in pituitary plus medium. However, this ratio was slightly but consistently higher in medium from the C3H/St strain, raising the interesting question whether such a preponderance prevails in the circulation of this and other mammary tumor-prone strains as well.

Extracts of mouse and rat adenohypophyses have been analyzed for a low-molecular-weight variant of growth hormone (GH) known to occur in humans, the so-called "20K"-GH (mol wt = 20,000 vs 22,000 for traditional human GH). Pituitary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostained with an antiserum raised against murine GH. Reactive and unreactive bands in close vicinity of the major GH band were fingerprinted by a peptide-mapping technique that reveals only the tyrosine-containing peptides, but can be applied to fingerprint single protein bands within gels. We found a protein band 2000 mol wt smaller than the major murine GH in both mouse and rat pituitary glands whose fingerprint resembled that of major GH. The peptide-mapping results are consistent with the interpretation that the internal deletion of amino acid residues most likely is in the same region of the molecule as in the human 20K-GH. Unlike the human 20K-GH, however, the murine counterpart showed no cross-reactivity with an antiserum raised against 22K-GH in the test system used here.

The effects of different doses of testosterone on basal and stimulated secretion of prolactin (PRL) were investigated. Intact female mice of the S/W strain were injected sc with 0, 1, 10, 20, 50, 250, 500, 1000, 2500, or 5000 micrograms of testosterone propionate (TP) once daily for 4 weeks. Serum testosterone concentrations of TP injected mice rose 2- to 5-fold above those of controls at 1- to 50-micrograms doses, and 25- to 600-fold over controls in mice given higher doses of the steroid. Administration of 1 microgram of TP, the lowest dose tested, had no significant effects on basal serum PRL concentrations and only slightly inhibited the release of PRL induced by perphenazine, but the weight and PRL concentration of the pituitary gland were significantly depressed. However, at doses of 10, 20, and 50 micrograms TP, perphenazine-induced PRL release, pituitary PRL concentration, and pituitary gland weight were all reduced in a dose-related manner. The basal serum PRL concentrations decreased by the time the dose of TP reached 50 micrograms. In contrast, higher doses of TP (250 micrograms and above) reversed the suppression of these parameters: pituitary gland weight and basal serum PRL levels were restored to control levels, whereas pituitary PRL concentrations and perphenazine-induced PRL release were partially restored. These results suggest that administration of moderate and large doses of testosterone may suppress natural episodic and acute releases of PRL.

The intra-lymph node technique used to inoculate rabbits with small quantities of antigen has been described. A variety of antigens in the 20,000-22,000 molecular weight range, as well as a 15-amino acid peptide coupled to BSA, have been inoculated successfully by this procedure. We have made no attempt to compare the success rate of the intra-lymph node inoculation route with other techniques utilizing small (microgram) quantities of antigen.

We investigated the effects of high doses of estrogen on the basal and stimulated secretion of PRL. Daily administration of 0.1 micrograms estradiol benzoate to female mice for 4 weeks significantly increased the amplitude of PRL release induced by perphenazine. A dose of 1.0 micrograms had no stimulatory effect, while doses of 10, 20, and 50 micrograms completely prevented the release of PRL in response to perphenazine. The rise in basal serum PRL concentrations seen at lower doses of estradiol benzoate was also minimized with 20- and 50-micrigrams doses. At the same time, PRL concentrations within the pituitary gland as well as pituitary weight consistently increased in response to all high doses of estradiol benzoate tested, although the increment at the highest dose (50 micrograms) was somewhat smaller. These results suggest that prolonged administration of estrogen in large amounts may be detrimental to the natural, acute release of PRL. We postulate that this effect may account, in part, for the observed mammostatic and antigalactic effects of high doses of estrogen.

The effect of lowering PRL levels in blood during early infancy on subsequent growth and development was studied in mice. PRL was reduced by injecting either an antiserum raised against homologous PRL or a PRL-inhibiting drug, 2-chloro-6-methylergoline-8 beta-acetonitrile methanesulfonate (ergoline), into 4-day-old mice for a period of 4 or 5 days. Both the anti-PRL serum and ergoline rapidly killed some of the injected animals, but the effect of anti-PRL serum was much more severe than that of ergoline (39% vs. 8.7% mortality during the period of injection). Similar administration of an antiserum against mouse GH or the GH-inhibiting peptide somatostatin did not cause a significant number of deaths. The deaths from the anti-PRL serum largely ceased when the antiserum was neutralized with rat PRL (NIH-RP-1) before injection. The gain in body weight of baby mice was markedly retarded within 24 h of injecting anti-PRL serum and ergoline, in contrast to the anti-GH serum and somatostatin injections, which took 3--4 days to inhibit growth perceptibly. The anti-PRL serum, despite having only one eighth the titer of anti-GH serum, was by far the most effective of the two antisera in diminishing tibial epiphyseal cartilage width as well as weights of pituitary glands, testes, and adrenals and retarding sexual maturity. The more severe and generalized developmental abnormalities and the incidence of mortality as a result of anti-PRL serum administration suggest that PRL in mice may be involved in the maintenance of some vital function during infancy.

Snell (dw/dw) and Ames (df/df) dwarf mice of both sexes were evaluated for immunoassayable PRL in plasma and for the presence of PRL-containing cells in the hypophysis. Regardless of the method of blood collection (decapitation or cardiac or orbital puncture), minimal concentrations of PRL were detected in the plasma of hereditary dwarf mice. PRL secretion was not augmented in Snell or Ames female dwarfs after treatment with perphenazine or estradiol benzoate, stimuli which greatly increased PRL release in normal female littermates. Comparison of PRL levels in dwarf animals (dw/dw or df/df) using two different homologous RIAs substantiated the observation that male and female dwarfs are PRL deficient. Mammotropes, readily detectable in the pituitary glands of all normal siblings of Snell and Ames mice, were absent from the dwarf mouse hypophysis, which was markedly reduced in size. The lack of PRL-containing cells in the dwarf mouse pituitary may explain why peripheral PRL levels in this animal are below those measured in hypophysectomized mice.

The influences of castration and of subsequent replacement therapy with gonadal hormones on the secretion of prolactin (PRL) and growth hormone (GH) in male and female mice were investigated. Long term castration in female mice significantly reduced basal and perphenazine-indueed PRL levels in the serum and PRL cOncentrations in the pituitary gland. Treatment with a moderate dose (1 pg/day) of estradiol benzoate (EB) for several weeks increased PRL levels in the pituitary gland and serum, but the release of PRL in response to perphenazine was not augmented. A large dose of EB(20�sg/day), on the other hand, resulted in a smaller increase in pituitary concentrations, little or no increase in basal serum levels and complete abolition of perphenazine-induced PRL release, suggesting that high doses of EB are detrimental to the release of PRL. In male mice, castration had little or no effect on PRL levels in the pituitary gland or on basal concentrations in serum, but it significantly reduced perphenazine-induced release of PRL. Administration of testosterone (0.5 mg/day) only marginally enhanced pituitary and serum concentrations of PRL, whereas EB injection (1 11g/day) increased serum and pituitary PRL concentrations severalfold. However, neither testosterone nor EB treatment of orehideetomized males could cause serum PRL concentrations to rise after injection of perphenazine to levels equivalent to those in intact females. This suggests that the sex difference in the control of PRL secretion in adult mice involves the pituitary-hypothalamic system rather than the gonad. Concentrations of GH in the pituitary glands and sera of female mice increased following castration, whereas administration of EB (1 pg/day) decreased basal levels of GH in serum and prevented the usual posteastration rise in serum and pituitary GH. In male mice, in contrast, castration reduced pituitary GH concentrations but had no marked influence on serum GH. Administration of testosterone (0.5 mg/day), in turn, increased pituitary GH concentrations, with no appreciable influence on serum GH. These results indicate that the female gonad exerts a net inhibitory influence on GH secretion in female mice, whereas, although essential for maintaining normal levels in the pituitary gland, the male gonad has little influence on the circulating levels of GH in male mice.

Journal Article Hormone Production by the Pituitary and Testes of Male C57BL/6J Mice During Aging Get access C. E. FINCH, C. E. FINCH ∥Andrus Gerontology Center and Department of Biological Sciences, University of Southern California Los Angeles, California 90007 Search for other works by this author on: Oxford Academic Google Scholar V. JONEC, V. JONEC *VA Hospital, Sepulveda California 91343 Search for other works by this author on: Oxford Academic Google Scholar J. R. WISNER, JR., J. R. WISNER, JR. ∥Andrus Gerontology Center and Department of Biological Sciences, University of Southern California Los Angeles, California 90007 Search for other works by this author on: Oxford Academic Google Scholar Y. N. SINHA, Y. N. SINHA †Division of Diabetes and Endocrinology, Scripps Clinic and Research Foundation Lajolla, California 92037 Search for other works by this author on: Oxford Academic Google Scholar J. S. DE VELLIS, J. S. DE VELLIS ‡Department of Anatomy, University of California Los Angeles Los Angeles, California 90024 Search for other works by this author on: Oxford Academic Google Scholar R. S. SWERDLOFF R. S. SWERDLOFF §Department of Medicine (Endocrinology), UCLA School of Medicine, Harbor General Hospital Campus Torrance, California 90502 Search for other works by this author on: Oxford Academic Google Scholar Endocrinology, Volume 101, Issue 4, 1 October 1977, Pages 1310–1317, https://doi.org/10.1210/endo-101-4-1310 Published: 01 October 1977 Article history Received: 01 October 1976 Published: 01 October 1977