Wesley C. Hymer

Characterization of Estrogen-Induced Adenohypophyseal Tumors in the Fischer 344 Rat

Pituitary tumors were induced in F344 female rats by chronic treatment with diethylstilbestrol (DES, 8-10 mg) implanted subcutaneously in silastic capsules. Over a range of 1-150 days of DES treatment, pairs of control and DES-treated rats were sacrificed, and their pituitaries dissociated enzymatically into single-cell preparations. The cell populations were examined regarding total cell recovery correlated with gland weight, intracellular prolactin (PRL) content and subsequent release in primary culture, immunocytochemical PRL staining, density and/or size alterations via separation on Ficoll-Hypaque and by unit gravity sedimentation, and cell cycle analysis, after acriflavine DNA staining, by laser flow cytometry. Total cell yields from DES-treated pituitaries increased from 1.3 times control yields at 8 days of treatment to 58.9 times control values by day 150. Intracellular PRL content ranged from 1.9 to 9.4 times control levels, and PRL release in vitro was significantly and consistently higher than controls, after at least 8 days of DES exposure. Beyond 8 days of DES exposure, the immunochemically PRL-positive proportion of cells increased to over 50% of the total population. Increased density and/or size and PRL content were indicated for the majority of the PRL cell population in both types of separation protocols. All these effects of DES were more pronounced among previously ovariectomized animals. The data extend the findings of other investigators, further establishing the DES-induced tumor as a model for study of PRL cellular control mechanisms.

High-affinity growth hormone binding protein and acute heavy resistance exercise

PURPOSE The purpose of this investigation was to examine the influence of resistance training on circulating concentrations of growth hormone binding protein (GHBP) in response to acute heavy resistance exercise. METHODS Using a cross-sectional experimental design, a group of resistance-trained men (RT, N=9, 7.9+/-1.3 yr resistance training experience) and a group of untrained men (UT, N=10) performed an acute heavy resistance exercise protocol (AHREP) consisting of 6 sets of 10 repetition maximum parallel squats. Blood samples were obtained 72 h before exercise, immediately before exercise, and 0, 15, 30, 45, and 60 min after exercise. RESULTS Significant increases (P<0.05) in GHBP, immunoreactive growth hormone (iGH), and IGF-1 were observed in both subject groups after AHREP. There were no differences (P>0.05) between groups in GHBP at rest or after AHREP. However, RT exhibited a significantly greater iGH response to AHREP than UT subjects, and significantly higher IGF-1 values at rest and after exercise. Significant positive correlations were found between GHBP and BMI, body fat, and leptin in both groups. A significant positive correlation also was observed between resting leptin and GHBP values in UT but not RT subjects. CONCLUSIONS In summary, these data indicate that resistance training does not increase blood GHBP. Nevertheless, the increases observed with IGF-1 concentrations in the resistance-trained subjects do suggest an apparent adaptation with the regulation of this hormone. If there was in fact an increase in GH sensitivity and GH receptor expression at the liver that was not detected by blood GHBP in this study, it may be possible that factors contributing to the circulating concentration of GHBP other than hepatocytes (e.g., leptin and adipocytes) may serve to mask training-induced increases in circulating GHBP of a hepatic origin, thus masking any detectable increase in GH receptor expression.

Pituitary hollow fiber units in vivo and in vitro.

A new technique is described for the study of mammalian pituitary cells in vivo and in vitro. The method involves encapsulation of freshly trypsinized rat, sheep or human pituitary cells in XM-50 Amicon hollow fibers followed by their intracranial implantation into hypophysectomized rats. These pituitary fiber units promoted recipient growth for 3 weeks before weight gains plateaued. Body composition analyses showed that significant quantities of protein, fat and ash accounted for the weight gain. Morphological study of the capsule contents 7-39 days postimplantation revealed the presence of intact somatotrophs and corticotrophs. The hollow fibers may provide an immunologically privileged site by virtue of the fact that the 50,000 dalton pores making up the lumen surface permit pituitary hormones to diffuse from the capsule, but theoretically do not permit immunoglobulin molecules to penetrate the capsule. Growth of hypox rats receiving capsules containing allogeneic rat pituitary cells, sheep cells or pieces of human postmortem pituitary support this concept. Furthermore, rats implanted with human PRL adenoma cells had detectable quantities of circulating hPRL 100 days postimplantation. It is suggested that the pituitary hollow fiber units function when they come in contact with a ventricular surface of a hypox animal. With these units, it will be possible to study function of the same group of pituitary cells in vitro and in vivo.

Growth hormone molecular heterogeneity and exercise

NINDL, B. C., W. J. KRAEMER, J. O. MARX, A. P. TUCKOW, and W. C. HYMER. Growth hormone molecular heterogeneity and exercise. Exerc. Sport Sci. Rev., Vol. 31, No. 4, pp. 161–166, 2003. There are more than 100 molecular isoforms of circulating growth hormone (GH), but the traditional measurement approach in the exercise literature has only focused on the main isoform ( i.e., 22 kDa). New assay methodologies now can assess various GH isoforms. The current data suggest that exercise results in the preferential release of GH isoforms with extended half-lives, thereby sustaining biological actions.

Effects of estrogen-induced hyperprolactinemia on endocrine and sexual functions in adult male rats

Chronic estrogen treatment can lead to development of prolactin (PRL) secreting pituitary tumors. We have tested the ability of diethylstilbestrol (DES) to produce persistent hyperprolactinemia (hyperPRL) in adult male rats and examined the effects of this treatment on hypothalamic-pituitary-testicular function, adenohypophyseal structure, copulatory behavior and fertility. Silastic capsules containing approximately 5 mg DES were subcutaneously implanted into adult male CDF (F-344)/CrlBR rats and removed 15 or 20 weeks later. Extreme hyperPRL, as well as suppression of plasma LH and FSH levels, persisted after DES capsules were removed. In contrast, plasma testosterone levels increased rapidly after removal of DES capsules and reached normal levels within 4-6 weeks. Copulatory behavior was assessed on two occasions between 7 and 14 weeks after removal of the DES capsules and was found to be suppressed in DES-treated rats, as evidenced by significant increases in latencies to mount, to intromit and to ejaculate. Moreover, when the animals were placed with normal females, the interval until conception was significantly greater in DES-treated than in control males. In spite of these differences in copulatory behavior, 10 of 11 DES-treated males were fertile. At autopsy, 44 weeks after capsule implantation (i.e. 24 or 29 weeks after capsule removal), DES-treated rats had marked enlargement of the anterior pituitary, increased weights of the lateral prostate and the adrenals, increased levels of testicular hCG-binding sites, reduced concentration of dopamine and norepinephrine in the median eminence and increased concentration of LHRH in the preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture of human pituitary prolactin and growth hormone cells

SummaryFragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues.

A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide × 1.5 mm thick in the laboratory version and 16 cm wide × 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions.Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.

Separation of cells from the rat anterior pituitary gland

Publisher Summary This chapter reveals that the rat anterior pituitary gland contains six hormone-producing cell types hence, cell separation techniques can be used to obtain enriched populations of these different cell types. In turn, these separated cells are useful reagents for mechanism of action studies to investigate intracellular events leading up to hormone release. The chapter reviews the pituitary cell separation. The purity, viability, and responsiveness of the separated cells will depend upon (a) the physiological state of the pituitary donor, (b) the tissue dissociation procedure, (c) the staining technique used for identification of cell type, and (d) the cell separation technique employed. A majority of the hormone contained within a pituitary cell is stored in secretion granules. These granules are of varying size and density; as such, they affect the sedimentation properties of the parent cell. For example, GH cells from 2-week-old rats are only sparsely granulated whereas after 8 weeks GH cells are the most dense of the total cell population.

DNA Synthesis in the Anterior Pituitary of the Male Rat: Effect of Castration and Photoperiod

Castration increased incorporation of tritiated thymidine into total DNA in the anterior pituitary gland. Furthermore, there was a threefold increase in the percentage of labeled basophils 1 month after castration. Exposure of rats to constant light or dark also changed DNA synthesis; these changes depended on age of the animal and on exposure length. The results reflect physiologically induced mitotic activity in specific classes of pituitary cells and further suggest that neuroendocrine mechanisms may be involved in control of cell turnover in the gland.

DNA synthesis in adult rat anterior pituitary glands in organ culture

Abstract Anterior pituitary glands of adult male rats weighing 175–500 g were cultured for periods up to 13 h in Medium 199 containing 2 μc/ml of 3H-thymidine. Extraction of the nucleic acids from pituitary homogenates by conventional biochemical procedures indicated that the 3H-thymidine was incorporated into DNA. In addition, radioautographs showed that the newly synthesized DNA was nuclear and in pituitary parenchymal cells. Labeled mitotic figures were found, and labeled acidophils and basophils were also observed. These results indicate that pituitary cells committed to the synthesis of specific hormones can also synthesize DNA.