Pituitary Fractions Research Articles

Association of Gnrhr mRNA With the Stem Cell Determinant Musashi: A Mechanism for Leptin-Mediated Modulation of GnRHR Expression.

The cyclic expression of pituitary gonadotropin-releasing hormone receptors (GnRHRs) may be an important checkpoint for leptin regulatory signals. Gonadotrope Lepr-null mice have reduced GnRHR levels, suggesting these receptors may be leptin targets. To determine if leptin stimulated GnRHR directly, primary pituitary cultures or pieces were exposed to 1 to 100 nM leptin. Leptin increased GnRHR protein levels and the percentages of gonadotropes that bound biotinylated analogs of gonadotropin-releasing hormone (bio-GnRH) but had no effect on Gnrhr messenger RNA (mRNA). An in silico analysis revealed three consensus Musashi (MSI) binding elements (MBEs) for this translational control protein in the 3' untranslated region (UTR) of Gnrhr mRNA. Several experiments determined that these Gnrhr mRNA MBE were active: (1) RNA electrophoretic mobility shift assay analyses showed that MSI1 specifically bound Gnrhr mRNA 3'-UTR; (2) RNA immunoprecipitation of pituitary fractions with MSI1 antibody pulled down a complex enriched in endogenous MSI protein and endogenous Gnrhr mRNA; and (3) fluorescence reporter assays showed that MSI1 repressed translation of the reporter coupled to the Gnrhr 3'-UTR. In vitro, leptin stimulation of pituitary pieces reduced Msi1 mRNA in female pituitaries, and leptin stimulation of pituitary cultures reduced MSI1 proteins selectively in gonadotropes identified by binding to bio-GnRH. These findings show that leptin's direct stimulatory actions on gonadotrope GnRHR correlate with a direct inhibition of expression of the posttranscriptional regulator MSI1. We also show MSI1 interaction with the 3'-UTR of Gnrhr mRNA. These findings now open the door to future studies of leptin-modulated posttranscriptional pathways.

Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.

Parathyroid hormone-related peptide directly increases adrenocorticotropic hormone secretion from the anterior pituitary in hens

The presence of the receptor for parathyroid hormone-related peptide (PTHrP) and the effect of PTHrP on adrenocorticotropic hormone (ACTH) secretion in the hen anterior pituitary were examined. The plasma membrane fraction of the anterior pituitary was found to contain a specific chicken PTHrP (cPTHrP) binding component. The binding component had properties of a receptor, such as binding specificity to cPTHrP, reversibility, saturable binding, high affinity, and limited capacity; therefore, it was elucidated that the PTHrP receptor exists in the plasma membrane of the hen anterior pituitary. A third ventricular injection of cPTHrP in nonlaying hens caused a decrease in the chicken ACTH level of the anterior pituitary and an increase in the chicken ACTH level of blood plasma, with an increase in the binding affinity and a decrease in the binding capacity of PTHrP receptor in the anterior pituitary. The present study suggests that PTHrP may act directly on the anterior pituitary via its receptor binding and may enhance ACTH secretion from this tissue in hens.

Effects of Pituitary Glycoprotein Hormones and Thyroid Hormones on In-Vitro Vitellogenin Incorporation into Organ-Cultured Oocytes in the Japanese Eel, Anguilla japonica

The objectives of the present study were to establish a long-term culture system for previtellogenic ovarian fragments of the Japanese eel (Anguilla japonica) and to identify the effects of salmon pituitary glycoprotein fraction (SPG), thyroxine (T4), and 3,5,3'-triiodothyronine (T3) on the uptake of vitellogenin (VTG) by cultured ovarian fragments evidenced by the appearance of yolk globules (YGs) within the oocytes. Yolk globules first appeared in the oocytes incubated in media containing only VTG (VTG-only group) after 9 days, whereas YGs began to accumulate in the oocytes of ovarian fragments cultured in media containing VTG+SPG (SPG group) following only 3 days of incubation. Furthermore, the occurrence of vitellogenic oocytes (%VO) and proportion of YGs within oocytes (%YG area) were significantly higher in follicles cultured in 30 ng/ml SPG throughout the culture period. No such stimulatory effects of T4 on VTG uptake were observed. Incubation of ovarian fragments with VTG and T3 (T3 group; 50 ng/ml) resulted in an increased %VO compared to follicles in the VTG group by day 9 of culture, and from day 10 onwards, both %VO and %YG area became significantly higher in follicles of the T3 group. Interestingly, SPG stimulated VTG incorporation and YG accumulation even in small oocytes (approximately 150 microm), whereas T3 showed these effects only in larger sized oocytes (> 180 microm). These results suggest that both SPG and T3 can accelerate VTG incorporation, but the mechanisms whereby this is achieved may differ between these hormone preparations.

Immunocytochemical localization of secretory phospholipase A(2)-like protein in the pituitary gland and surrounding tissue of the bullfrog, Rana catesbeiana.

Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A(2) (PLA(2)) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA(2)-like protein were observed predominately in these secretory granules. These findings support the view that this PLA(2)-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631-637, 2001)

Prolactin kinase activity in bovine anterior pituitary sub-cellular fractions

Bovine anterior pituitary cells phosphorylate prolactin (PRL). We describe the phosphorylation of endogenous and exogenous bPRL in highly enriched subcellular fractions of bovine anterior pituitary using [ γ- 32P]-ATP. 32P-labeling of endogenous and exogenous bPRL occurred in all subcellular membrane fractions, but most significantly in the fraction enriched for secretory granules. Zn 2+ (0.8 mM), Cu 2+ (0.8 mM), and Mn 2+ (9.8 mM) increased bPRL phosphorylation by 268, 214, and 154%, respectively, relative to basal phosphorylation with no added cations. Neither Mg 2+ (10 mM) nor Ca 2+ (0.9 mM) increased bPRL phosphorylation above basal levels. Phosphorylation was dependent on the concentration of Zn 2+ with an apparent K m of 570 μM. bPRL phosphorylation occurred over a wide pH range of 5.9–8.3, with the greatest activity at pH of 6.7 or greater. Phosphorylation of bPRL was time-dependent. The apparent K ms of the bPRL kinase for exogenous bPRL and ATP were 15.3 and 267 μM, respectively. bPRL incorporation of 32P was unaffected by the presence of calcium and calmodulin, cAMP, phosphotidylserine and diolein, or spermine. From these results we conclude that in vitro phosphorylation of bPRL occurs under physiological conditions that would be found in pituitary cells.

GH kinase activity in bovine anterior pituitary subcellular fractions.

Growth hormone (GH) and prolactin (PRL) share significant structural homology. We have previously characterized the phosphorylation of bovine PRL and wish to determine whether a similar kinase activity phosphorylates bovine GH. Phosphorylation of bovine GH was performed using [alpha-32P]ATP labeling of subcellular fractions. Bovine GH phosphorylation was dependent on Zn2+ or Cu2+ with apparent Km's of 0.9 and 1.0 mM, respectively, and a pH maxima of 7.0. The apparent Km's of bovine GH kinase activity for exogenous bovine GH and ATP were 30 microM and 376 microM, respectively. Exogenous bovine PRL served as a competitive substrate, increasing the apparent Km for bovine GH by threefold compared to the Km determined without exogenous bovine PRL. We conclude: 1) in vitro phosphorylation of bovine GH occurs under conditions that are consistent with those found in anterior pituitary cells, and 2) a similar kinase activity phosphorylates both bovine PRL and GH.

A novel method of hormone administration for inducing gonadal maturation in fish

A new water-in-oil-in-water (W/O/W) type emulsion using lipophilized gelatin (LG) with palmitic anhydride and cotton-seed oil was developed for a novel method of hormone administration in fish. In the in vitro release experiments, the concentration of LG in the emulsion was determined to be 2% and the volume ratio of LG solution to cotton-seed oil adequate for obtaining the best-formed emulsion was 1:2. Plasma profiles of salmon GtH II were monitored in the Japanese eel during in vivo release experiment. Groups administered LG emulsion containing either pituitary saline extract or glycoprotein fraction of salmon pituitary had plasma salmon GtH II levels that increased slowly, peaked after 24 h (about 2–2.5 μg ml −1) and then gradually decreased. In the group administered only salmon pituitary saline extract, the levels rapidly attained a very high peak (about 20 μg ml −1) after 3 h and then acutely decreased. These results clearly indicate that GtH is gradually released from the LG emulsion.

Detection of Bovine Pituitary Opioid Precursors During Gel Permeation Using Trypsin and Cyanogen Bromide Treatment Followed by Radioimmunoassay

Abstract An enzymatic-plus-chemical treatment of fraction aliquots has been developed to monitor the presence of opioid peptides and opioid-containing precursors in chromatographic fractions. Aliquots of chromatographic fractions of bovine pituitary are treated with trypsin and CNBr. Homoserine lactone enkephalin is produced in those fractions that contained proenkephalin A- or proopiomelanocortin(POMC)-derived opioid-containing precursors. A radioimmunoassay (RIA) using an antibody that recognizes homoserine lactone enkephalin is used as the detector to identify the opioid precursor fractions. These fractions can be further characterized by mass spectrometry (MS) techniques.

Properties of Vasoactive Intestinal Polypeptide Receptors in the Anterior Pituitary of Female Chickens.

The membrane fraction of anterior pituitary in laying hens was found to possess a vasoactive intestinal polypeptide (VIP)-binding component having the properties of a receptor. Scatchard analysis of saturation studies revealed that the binding component had a single class of binding site with an equilibrium dissociation constant (Kd) of 0.092 ± 0.008 (mean ± SEM; n=5) nM and the maximum binding capacity (Bmax) of 631 ± 41 fmol/mg protein. The Kd value determined from kinetic analysis (0.091 ± 0.002 nM; n=3) was very similar to that obtained from Scatchard analysis. The Kd value obtained from Scatchard analysis was not significantly different between the laying and nonlaying hens. The Bmax in laying hens was greater than that in nonlaying hens. The administration of chicken VIP to nonlaying hens caused a marked decrease in the VIP receptor binding in the anterior pituitary with a concomitant increase in the blood serum concentrations of prolactin. The results may provide an evidence for a direct action of VIP on the anterior pituitary of female chickens.

Identification and localization of 23,000 and glycosylated rat prolactin in subcellular fractions of rat anterior pituitary and purified secretory granules.

Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting. The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice-cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane-bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane-bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants. The systematical occurrence of the array of near Mr glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.

Ovine luteinizing hormone: isoforms in the pituitary during the follicular and luteal phases of the estrous cycle and during anestrus.

Pituitaries were collected from late follicular phase (n = 5), mid-luteal phase (n = 5), and anestrous ewes (n = 4) to assess changes in intrapituitary LH heterogeneity at selected reproductive states. After homogenization, an aliquot of each pituitary extract was desalted by flow dialysis against water and chromtofocused on a pH 10.5 to 4.0 gradient. Concentrations of LH in pituitary extracts and chromatofocusing fractions were determined by RIA. The LH in pituitary extracts resolved into 13 isoforms during chromatofocusing, which were coded with letters beginning with the most basic isoform. Follicular and mid-luteal phase ewes exhibited similar distributions of intrapituitary LH among its isoforms. Relative to follicular and luteal phase ewes, anestrous ewes had lower percentages of isoforms D and E as well as higher percentages of isoforms G, H, J and K. Isoform F, the predominant molecular form of LH, constituted a similar percentage in all treatment groups (P > .05). Thus, the distribution of intrapituitary LH among its isoforms did not change significantly between the mid-luteal and follicular phases of the estrous cycle, but higher percentages of the weakly basic and acidic forms of LH were present during anestrus. These observations suggest that intrapituitary LH heterogeneity changes minimally throughout the estrous cycle of ewes during the breeding season.

Maturation of follicle-stimulating hormone gonadotropes during the rat estrous cycle.

FSH mRNA is transcribed after the onset of high FSH secretion during proestrus and estrus. Pituitary cell fractions separated by size and density were studied to determine if expression of FSH mRNA activity was predominantly in one subset during the estrous cycle and to determine the source and significance of "silent FSH" cells that secrete FSH, but store too little for detection. Pituitary cells were separated by centrifugal elutriation, plated, and then exposed to 0.1-1 nM [D-Lys6]GnRH for 3 h. Media were assayed for FSH by RIA, and the cells were fixed for immunocytochemistry or in situ hybridization. The percentages of immunoreactive FSH cells in unseparated populations increased from 8% at metestrus to 12% during proestrus. Percentages of cells with FSH beta mRNA showed the same rise; however, peak levels were higher (17%) during proestrus and estrus. Small cells with FSH beta mRNA were more frequent than those with antigens early in the cycle. The largest cell fractions contained 38-44% immunoreactive cells. Only 8-21% of these cells had FSH beta mRNA, except during the morning of proestrus (33%). The distribution analyses showed that the increment in immunoreactive FSH cells during diestrus initially stemmed from smaller subsets; however, over half of immunoreactive FSH cells were large by the evening of proestrus. During the time of active transcription of FSH mRNA, more than half of the cells with FSH beta mRNA were small or medium-sized. Thus, early in the cycle, FSH beta mRNA is transcribed in the smaller cells, which may be the source of the silent FSH cells reported in previous studies. During proestrus, smaller FSH cells also secreted as well if not better than those in the unseparated population or large fractions. When they secreted more than expected from their percentages of FSH cells, this response was interpreted to be due to either the presence of cells that are immunoreactively silent or the possible removal of autocrine or paracrine regulatory factors.

SIIp: a unique secretogranin/chromogranin of the pituitary released in response to gonadotropin-releasing hormone.

A monoclonal antibody prepared by immunization of mice with a rat pituitary granule fraction stained a single band on a Western blot of pituitary homogenate (bovine, ovine, porcine, or rat) with an apparent mol wt of 78,000 (7.5% acrylamide gel in sodium dodecyl sulfate) and pI 5.0-5.1 (isoelectric focusing). Subcellular fractionation studies of rat pituitaries indicated that the determinant of the monoclonal antibody was markedly enriched in the secretory granule fraction, an observation that was independently confirmed by immunohistochemistry of intact cells. Immunohistochemistry also indicated that this determinant was selectively located in gonadotropes and thyrotropes. On Western blots, this band comigrated with adrenal secretogranin-II (SII; chromogranin-C), had the same N-terminal sequence (six amino acids), and was heat stable (95 C; 10 min). The pituitary protein containing the determinant for the monoclonal antibody could be precipitated by a polyclonal antibody prepared by immunization of rabbits with the C-terminal sequence of adrenal SII (triodecapeptide). Conversely, the monoclonal antibody precipitated the protein containing the determinant for the polyclonal antibody. While both the monoclonal and polyclonal antisera recognized the pituitary molecule, only the polyclonal antibody recognized SII from the adrenal. A RIA was established and used to assess the release pattern of this molecule from pituitary cell cultures. Release was stimulated by GnRH and blocked by a GnRH antagonist. Release was Ca2+ dependent and stimulated by either phorbol myristyl acetate (a protein kinase-C activator) or NaF (a G-protein activator). GHRH and TRH were not as effective secretogogues as GnRH. The observations that a unique form of SII is present in the pituitary gonadotrope and secreted in response to a specific endocrine stimulus present the possibility that this substance has an endocrine function. Further, the tissue specificity of the determinant suggest that it may be useful for the specific diagnosis and monitoring of pituitary tumors.

Effect of an essential fatty acid deficiency on the phospholipid composition in anterior pituitary membranes

The effects of an essential fatty acid deficient diet were investigated on the phospholipid fatty acids of several membrane fractions of the rat anterior pituitary, the secretion of which is known to be partly dependent on the membrane phospholipidic constituents. In standard dietary conditions, arachidonic acid (20:4n-6) and its elongation product, adrenic acid (22:4n-6), were the two main polyunsaturated fatty acids in all fractions studied. In rats deprived of EFA for 6 weeks after weaning, the levels of both 20:4n-6 and 22:4n-6 were not changed in microsomal + plasma membrane and nuclear fractions, whereas they were decreased in heavy mitochondrial and light mitochondrial fractions. The present data suggest a mechanism of compensation between membrane fractions which may preferentially preserve 20:4n-6 and 22:4n-6 in discrete membrane fractions.

Changes in Proopiomelanocortin Primary Transcript Levels in the Anterior Pituitary Accompany Increased Adrenocorticotropin Secretion During the Diurnal Surge

Proopiomelanocortin (POMC) gene transcription in the anterior pituitary varies during stress and glucocorticoid feedback. These changes appear to parallel alterations in peptide release. The diurnal rhythm of the hypothalamo-pituitary-adrenal axis also involves the periodic excursion of adrenocorticotropin (ACTH) levels in plasma, but it is not clear whether the diurnal release is accompanied by changes at the transcriptional level. In the present study, we have initially characterized the heteronuclear species of POMC (hnPOMC) RNA found in the anterior pituitary by a Northern blot analysis and subsequently used this method to quantitate relative changes in the levels of heteronuclear transcript during diurnal stimulation. Two species of RNA migrating at 6.0 kb and 4.1 kb were found in the nuclear fraction of the anterior pituitary. Successive probing by various POMC cRNAs indicated that the 6.0 kb fragment was the primary transcript and the 4.1 kb fragment corresponded to the intron A-containing processing intermediate of POMC. The nuclear species were quantitated after acute swim stress and during the diurnal ACTH secretion. Acute swim increased plasma ACTH levels by 243% after 30 min. This was paralleled by a 214% increase in the primary transcript RNA levels. Endogenous circadian stimulation in the evening produced a smaller rise of plasma ACTH (79%), and was accompanied by a 34% increase in POMC hnRNA levels. Nuclear processing intermediate (4.1 kb) and the mRNA levels did not vary during the evening. These results suggest that the diurnal mechanism transiently increases ACTH release as well as POMC gene transcription in the anterior pituitary. Release and transcription appear to be tightly coupled during circadian activation as well as during stress.

Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat.

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.

Influence of suckling on tubulin-dependent GTPase activity in the anterior pituitary lobe of the lactating rat

A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.

Heightened secretion by small and medium-sized luteinizing hormone (LH) gonadotropes late in the cycle suggests contributions to the LH surge or possible paracrine interactions.

LH secretion from pituitary cell fractions separated by centrifugal elutriation was compared to learn whether any contributed to the LH surge. After plating (1 h) and stimulation with 0-10 nM [D-Lys6]GnRH (4 h), some fractions secreted levels that were out of proportion to their enrichment. Large cells from proestrous morning rats (3-fold enriched) secreted 4.3 times more LH, and medium-sized fractions (1.5-fold enriched) secreted 2-3.4 times more LH than unseparated cells during estrus or metestrus. Normalized data (nanograms per 20,000 cells) showed that basal levels reflected the enrichment in the fractions. Data were also normalized to nanograms of LH per 1,000 LH gonadotropes to focus on LH cell activity. Basal secretion in unseparated cultures (4-6 ng/ml/1,000 LH cells) was lower than that in small or large LH cells during all stages except proestrus, when small gonadotropes became as active as those in unseparated cultures, and large gonadotropes secreted 2-3 times more LH. Basal secretion from medium-sized gonadotropes was comparable to that in unseparated cultures. [D-Lys6]GnRH-mediated secretion from unseparated, small, and large LH cells was comparable during most stages. However, during proestrus, large gonadotropes secreted 2.2 times more LH than unseparated counterparts. Stimulated medium-sized LH cells were 1.3-2.3 times more active in most stages than those in unseparated cultures and 1.75-2.8 times more active than those in large cell fractions (from proestrous PM to metestrus). Whereas this enhanced secretion late in proestrus suggests that medium-sized LH cells may support the LH surge, it also may reflect the removal of regulatory factors from cells in other fractions. Studies of autocrine or paracrine interactions with gonadotropes are needed to test this hypothesis.

Pituitary Luteinizing Hormone Isohormones - Changes in Profiles during the Menstrual Cycle and Receptor Binding Activities in Cynomolgus Monkeys (Macaca fascicularis).

Changes were demonstrated in isoelectric focusing (IEF) profiles of pituitary luteinizing hormone (LH) in the cynomolgus monkey during the menstrual cycle. The LH activity was determined through radioreceptorassay (RRA) and exhibited a heterogeneous IEF profile. Highly alkaline isohormones (pl ?? 8.6) were predominant in the pituitary collected from animals in the follicular phase. The IEF profile from periovulatory period animals showed a significant increase in relative amounts of lesser-alkaline (pl=8.3 and 7.5) and neutral (pl=7.0) isohormones. The IEF profile from luteal phase animals showed considerable LH activity from pH 8.3 to 9.1 with a narrow peak of LH at pH 7.5. Seven isohormone fractions (pl=7.0 to 11.5) were obtained from male monkeys. Serial dilution of the 7 isohormone fractions as well as human chorionic gonadotropin (hCG) and monkey pituitary fractions were tested in the RRA. There was a stepwise increase in the slopes of the dose-response curves with increasing pl values of the isohormones. The receptor-binding property of hCG and the highest alkaline isohormone is not affected by the presence or absence of NaCl, while the binding of cynomolgus monkey pituitary LH and other isohormones apparently decreases with the presence of NaCl. Receptor-binding properties of each LH-isohormone were analyzed.