Bovine Pituitary Cell Cultures Research Articles

Ovine anterior pituitary production of follistatin in vitro.

Rat pituitary cells express messenger RNA for the activin-binding protein, follistatin (FS), and rat and bovine pituitary cell cultures secrete FS into the medium. In the present study, a previously validated, heterologous RIA for ovine FS was employed to investigate FS synthesis, secretion, and regulation in cultures of ovine anterior pituitary cells. The validity of the RIA was confirmed by the finding that FS immunoreactivity in ovine pituitary cell culture-conditioned medium diluted in parallel with purified bovine FS, and fractionation of the conditioned medium resulted in the coelution of activin-binding activity with the FS immunoactivity. The concentration of endogenous ovine FS achieved in the culture medium (0.08-0.6 nM) was in the range over which bovine FS suppresses FSH secretion in these cultures (IC50 = 0.5 nM). To characterize the relationship between endogenous FS and FSH secretion, dispersed ovine pituitary cells were preincubated with 10% fetal bovine serum for 2 days, then cultured between days 2-5 in the presence of a chemically defined serum substitute. Under these conditions, FS was continuously secreted at a rate of 12.1 +/- 1.8 ng/10(6) cells.day (mean +/- SEM; n = 18), whereas FSH was secreted at 64 +/- 13 ng/10(6) cells.day (n = 7). The secretion of FS and FSH changed in a reciprocal way as culture conditions were altered either by maintaining exposure of the cells to fetal bovine serum or by plating the cells at a 6- to 10-fold higher seeding density. Under the latter circumstance, for instance, FS secretion during the 3-day test period decreased to 47 +/- 14% (n = 10) and FSH secretion increased to 137 +/- 6% (n = 6) of the respective values in cultures of dispersed cells. FS secretion was increased nearly 3-fold (P < 0.05) in a dose-dependent manner by continuous exposure of ovine pituitary cells between days 2-5 to recombinant human activin A (1-10 nM), which concomitantly increased FSH secretion. Recombinant human inhibin A (0.003-10 nM); the synthetic glucocorticoids, RU28362 and dexamethasone (each 1-100 nM); the sex steroids, testosterone (1-100 nM), 17 beta-estradiol (0.001-5 nM), and progesterone (4-2500 nM); and the vitamin A derivative, retinoic acid (0.3-32 microM), each inhibited FSH secretion from these cultures, but only the last agent significantly (P < 0.05) increased FS secretion. Inhibin prevented the stimulation of FSH secretion by activin A without affecting its stimulation of FS secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Position 2 and position 2/Ala15-substituted analogs of bovine growth hormone-releasing factor (bGRF) with enhanced metabolic stability and improved in vivo bioactivity.

In order to prepare GRF analogs with high activity in vivo, a strategy was undertaken to stabilize the peptide to dipeptidylpeptidase IV (DPP-IV), a protease found in plasma which inactivates native human and bovine GRF by cleavage of the Ala2-Asp3 bond. Replacement of the Ala2 residue with Ser, Thr, or Gly in [Leu27]bGRF(1-29)NH2 resulted in peptides greatly stabilized against proteolysis in plasma, but having low inherent GH-releasing activity when tested in bovine pituitary cell cultures. Replacement of Gly15 with Ala15 was marginally effective in improving the in vitro bioactivity of this group of peptides. When tested for GH-hormone release in steers, however, the Thr2,Ala15 analog was four times more potent than bGRF(1-44)NH2. Eleven additional analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series were synthesized and evaluated for metabolic stability in bovine plasma and for GH releasing activity in steers in vivo and in rat pituitary cells in vitro. Two compounds, [Val2,Ala15,Leu27]dGRF(1-29)NH2 and [Ile2,Ala15,Leu27]-bGRF(1-29)NH2, had increased GH-releasing activity in steers over that of [Thr2,Ala15,Leu27]-bGRF(1-29)NH2 and over a previously reported super-potent analog, [desNH2Tyr1,D-Ala2,Ala15]-hGRF(1-29)NH2.

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.

Steroids can modulate transdifferentiation of prolactin and growth hormone cells in bovine pituitary cultures

Fluctuations in the proportions of pituitary acidophils (cells that release GH and PRL, either separately or concurrently) have been correlated with dynamic changes in the steroid hormone milieu. Since modulation of these acidophilic subtypes can occur without appreciable alterations in the total number of acidophils, it has been proposed that GH- and PRL-secreting cells can actually transdifferentiate (gain or lose the ability to release GH or PRL). To test this hypothesis, we examined the effects of steroids on the proportions of cells that released GH, PRL, or both hormones (i.e. mammosomatotropes) in bovine pituitary cell cultures. Specifically, anterior pituitary cells from castrated males were cultured for 6 days in the absence (controls) or presence of 17 beta-estradiol, cortisol, or progesterone. Reverse hemolytic plaque assays revealed that 6.64 +/- 1.2% of all pituitary cells released GH, while 68.8 +/- 5.3% were PRL secretors in control cultures. Cortisol and progesterone induced an increment in the proportions of GH-secreting cells (10 nM cortisol, +11.7 +/- 2.4%; 1000 nM cortisol, +10.5 +/- 4.7%; 1000 nM progesterone, +2.87 +/- 1.5%) above control values while decreasing the relative abundance of cells that released PRL (10 nM cortisol, -7.6 +/- 1.7%; 1000 nM cortisol, -6.6 +/- 1.2%; 1000 nM progesterone, -5.5 +/- 1.3%) below control values. However, 17 beta-estradiol was ineffective in this regard at doses of 0.1-1000 nM. A more critical examination of the steroid-induced changes revealed that they were attributable to increases in the proportions of cells that released GH alone and both hormones simultaneously along with a concomitant decrease in the fraction that secreted only PRL. Two lines of evidence discount the possibility that these effects were due to selective cell proliferation. First, the mitotic rate of cultures (assessed by immunofluorescent detection of bromodeoxyuridine incorporation) was only about 1% of all cells in both control and steroid-treated cultures. Second, blocking cell proliferation by the addition of cytosine arabinoside (100 microM) did not inhibit the cortisol-induced augmentation of GH-releasing cells. Taken together, these results substantiate the hypothesis that acidophilic subpopulations are capable of transdifferentiation given an appropriate hormonal signal.

Exogenous Growth Hormone Inhibits Bovine but Not Murine Pituitary Growth Hormone Secretion in vitro: Evidence for a Direct Feedback of Growth Hormone on the Pituitary

To test the hypothesis that one aspect of growth hormone (GH) regulation involves a direct effect of GH on its own secretion at the level of the pituitary, we evaluated the effects of human GH on basal and GH-releasing factor (GRF)-induced GH secretion in rat and bovine pituitary cell cultures. Both 4 h (acute) and 24 h (chronic) exposure of bovine pituitary cells to exogenous human GH significantly (p less than 0.001) reduced GH secretion. Acute treatment resulted in a decrease in GH secretion from 579 +/- 24 to 500 +/- 17 ng/ml 4 h (mean +/- SEM, n = 24) while chronic treatment resulted in a GH decrease from 595 +/- 21 to 483 +/- 17 ng/ml/4 h. In contrast, human GH treatment of rat somatotropes had no effect on basal GH secretion. GRF-stimulated GH secretion was not affected by either acute or chronic GH exposure in either species. These results demonstrate that there are species-specific differences in the ability of GH to regulate its own secretion and provide evidence for a direct inhibitory effect of GH at the level of the bovine pituitary.