Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting

1990 ◽  
Vol 126 (2) ◽  
pp. 269-274 ◽  
Author(s):  
D. Wynick ◽  
R. Critchley ◽  
M. S. Venetikou ◽  
J. M. Burrin ◽  
S. R. Bloom
Keyword(s):  
Cell Surface ◽  
Cell Sorting ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Female Rats ◽  
Normal Female ◽  
Pituitary Cells ◽  
Female Rat ◽  
Fluorescence Activated Cell Sorting ◽  
Surface Labelling

ABSTRACT As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96·7 ±1·7 (s.e.m.)% and 98±1·0% respectively by immunocytochemistry, and to 95·8 ±1·1% and 97±0·8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 ±3·4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin. Journal of Endocrinology (1990) 126, 269–274

Flow cytometric analysis of functional anterior pituitary cells from female rats

1990 ◽  
Vol 126 (2) ◽  
pp. 261-NP ◽  
Author(s):  
D. Wynick ◽  
M. S. Venetikou ◽  
R. Critchley ◽  
J. M. Burrin ◽  
S. R. Bloom
Keyword(s):  
Cell Surface ◽  
Cell Size ◽  
Anterior Pituitary ◽  
Laser Light ◽  
Female Rats ◽  
Pituitary Cells ◽  
Light Scatter ◽  
Cell Sorter ◽  
Anterior Pituitary Cells ◽  
Surface Labelling

ABSTRACT Laser-light scatter signals generated from living cells provide useful information with regard to both cell size (forward-angle light scatter) and granularity (ninety-degree or perpendicular light scatter). By measuring angles of light scatter and fluorescence, a fluorescence-activated cell sorter is capable of analysing and sorting cells on the basis of their size, granularity and cell-surface fluorescence. Using an electronically programmable individual cell sorter we were able to analyse single, viable, dispersed anterior pituitary cells of the female rat on the basis of their laser light scatter characteristics. Two distinct populations of differing granularity were defined: 26±2·2% (mean ± s.e.m.) were more granular and 74±3·5% less granular. Acutely dispersed anterior pituitary cells were labelled with antibodies against four of the anterior pituitary hormones, and cell size and granularity were compared amongst the different hormonal cell types. Somatotrophs were the most granular cell type, gonadotrophs were the largest and corticotrophs the smallest, whilst lactotrophs were of intermediate size. Labelling was demonstrated to be dependent upon the secretory state of the cell. Hypothalamic stimulating factors increased cell-surface labelling, whilst dopamine and somatostatin decreased labelling. These changes compare favourably with published data obtained by immunocytochemistry. Using dual-colour fluorescence cell surface labelling we were unable to define a population of cells secreting both prolactin and growth hormone (mammosomatotrophs). Journal of Endocrinology (1990) 126, 261–268

scholarly journals Direct Stimulatory Effects of Oxytocin in Female Rat Gonadotrophs and Somatotrophs In Vitro: Comparison With Lactotrophs

Endocrinology ◽  
2014 ◽  
Vol 156 (2) ◽  
pp. 600-612 ◽  
Author(s):  
Arturo E. Gonzalez-Iglesias ◽  
Patrick A. Fletcher ◽  
José A. Arias-Cristancho ◽  
Ruth Cristancho-Gordo ◽  
Cleyde V. Helena ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Cell Types ◽  
Pituitary Hormones ◽  
Prolactin Secretion ◽  
Female Rats ◽  
Pituitary Cells ◽  
Female Rat ◽  
Dependent Manner ◽  
Rat Pituitary Cells

The peptide oxytocin (OT) is secreted by hypothalamic neurons and exerts numerous actions related to reproduction. OT stimulation of prolactin secretion in female rats is important during the estrous cycle, pregnancy, and lactation. Here we report that OT also stimulates transients of intracellular Ca2+ concentration in somatotrophs and gonadotrophs as well as the release of GH and LH in a dose-dependent manner with EC50 values that closely correspond to the ligand affinity of the OT receptor (OTR). Remarkably, the hormone-releasing effect of OT in these two cell types is 2 orders of magnitude more sensitive than that in lactotrophs. The specific OTR agonist [Thr4,Gly7]-oxytocin acutely stimulated the release of LH, GH, and prolactin from female rat pituitary cells in primary culture and increased intracellular Ca2+ concentration in gonadotrophs, somatotrophs, and lactotrophs. In these three cell types, the effects on hormone release and intracellular Ca2+ of both OT and [Thr4,Gly7]oxytocin were abolished by the specific OT receptor antagonist desGly-NH2-d(CH2)5[D-Tyr2,Thr4]OVT but not by the highly selective vasopressin V1a receptor antagonist, d(CH2)5[Tyr(Me)2,Dab5]AVP. Furthermore, 10 nM arginine vasopressin stimulated LH and GH release comparably with a dose of OT that was at least 10 times lower. Finally, the presence of the OTR-like immunoreactivity could be observed in all three cell types. Taken together, these results show that OT directly stimulates gonadotrophs, somatotrophs, and lactotrophs through OT receptors and suggest that OT signaling may serve to coordinate the release of different pituitary hormones during specific physiological conditions.

FLUORESCENCE ACTIVATED CELL SORTING OF FUNCTIONAL ANTERIOR PITUITARY CELLS

Endocrinology ◽  
1982 ◽  
Vol 110 (5) ◽  
pp. 1831-1833 ◽  
Author(s):  
MICHAEL O. THORNER ◽  
JOAO L. C. BORGES ◽  
MICHAEL J. CRONIN ◽  
DONALD A. KEEFER ◽  
DEBRA LEWIS ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Anterior Pituitary ◽  
Pituitary Cells ◽  
Fluorescence Activated Cell Sorting ◽  
Anterior Pituitary Cells

17β-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture

2012 ◽  
Vol 302 (10) ◽  
pp. E1189-E1197 ◽  
Author(s):  
Liliana d. V. Sosa ◽  
Silvina Gutiérrez ◽  
Juan P. Petiti ◽  
Claudia M. Palmeri ◽  
Iván D. Mascanfroni ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Anterior Pituitary ◽  
Pituitary Cell ◽  
Female Rats ◽  
Akt Pathway ◽  
Pituitary Cells ◽  
Female Rat ◽  
Anterior Pituitary Cell ◽  
Pathway Activation ◽  
17Β Estradiol

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17β-estradiol (E2, 10 nM) and its membrane-impermeable conjugated estradiol (E2-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E2, E2-BSA, TRH, and E2/TRH differentially increased the PRL secretion, the highest levels were achieved with E2-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E2 or E2-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E2-BSA, TRH, and E2/TRH and totally inhibited the PRL levels stimulated by E2-BSA/TRH, suggesting that the mER mediated the cooperative effect of E2 on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E2-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E2-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E2. These finding showed that E2 may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E2 in the pituitary.

Control of secretion in anterior pituitary cells--linking ion channels, messengers and exocytosis

1988 ◽  
Vol 139 (1) ◽  
pp. 287-316
Author(s):  
W. T. Mason ◽  
S. R. Rawlings ◽  
P. Cobbett ◽  
S. K. Sikdar ◽  
R. Zorec ◽  
...  
Keyword(s):  
Ion Channels ◽  
Intracellular Calcium ◽  
Anterior Pituitary ◽  
Hormone Secretion ◽  
Cell Types ◽  
Pituitary Cell ◽  
Pituitary Cells ◽  
Calcium Activity ◽  
Anterior Pituitary Cells ◽  
Voltage Dependent

Normal anterior pituitary cells, in their diversity and heterogeneity, provide a rich source of models for secretory function. However, until recently they have largely been neglected in favour of neoplastic, clonal tumour cell lines of pituitary origin, which have enabled a number of studies on supposedly homogeneous cell types. Because many of these lines appear to lack key peptide and neurotransmitter receptors, as well as being degranulated with accompanying abnormal levels of secretion, we have developed a range of normal primary anterior pituitary cell cultures using dispersion and enrichment techniques. By studying lactotrophs, somatotrophs and gonadotrophs we have revealed a number of possible transduction mechanisms by which receptors for hypothalamic peptides and neurotransmitters may control secretion. In particular, the transduction events controlling secretion from pituitary cells may differ fundamentally from those found in other cell types. Patch-clamp recordings in these various pituitary cell preparations have revealed substantial populations of voltage-dependent Na+, Ca2+ and K+ channels which may support action potentials in these cells. Although activation of these channels may gate Ca2+ entry to the cells under some conditions, our evidence taken with that of other laboratories suggests that peptide-receptor interactions leading to hormone secretion occur independently of significant membrane depolarization. Rather, secretion of hormone and rises in intracellular calcium measured with new probes for intracellular calcium activity, can occur in response to hypothalamic peptide activation in the absence of substantial changes in membrane potential. These changes in intracellular calcium activity almost certainly depend on both intracellular and extracellular calcium sources. In addition, strong evidence of a role for multiple intracellular receptors and modulators in the secretory event suggests we should consider the plasma membrane channels important for regulation of hormone secretion to be predominantly agonist-activated, rather than of the more conventional voltage-dependent type. Likewise, evidence from new methods for recording single ion channels suggests the existence of intracellular sites for channel modulation, implying they too may play an important role in secretory regulation. We shall consider new data and new technology which we hope will provide key answers to the many intriguing questions surrounding the control of pituitary hormone secretion. We shall highlight our work with recordings of single ion channels activated by peptides, and recent experiments using imaging of intracellular ionized free calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia

Development ◽  
2002 ◽  
Vol 129 (18) ◽  
pp. 4229-4239 ◽  
Author(s):  
Lori T. Raetzman ◽  
Robert Ward ◽  
Sally A. Camper
Keyword(s):  
Cell Survival ◽  
Anterior Pituitary ◽  
Molecular Genetic ◽  
Cell Types ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Temporal Shift ◽  
Pituitary Development ◽  
Show Evidence

Deficiencies in the homeobox transcription factors LHX4 and PROP1 cause pituitary hormone deficiency in both humans and mice. Lhx4 and Prop1 mutants exhibit severe anterior pituitary hypoplasia resulting from limited differentiation and expansion of most specialized cell types. Little is known about the mechanism through which these genes promote pituitary development. In this study we determined that the hypoplasia in Lhx4 mutants results from increased cell death and that the reduced differentiation is attributable to a temporal shift in Lhx3 activation. In contrast, Prop1 mutants exhibit normal cell proliferation and cell survival but show evidence of defective dorsal-ventral patterning. Molecular genetic analyses reveal that Lhx4 and Prop1 have overlapping functions in early pituitary development. Double mutants exhibit delayed corticotrope specification and complete failure of all other anterior pituitary cell types to differentiate. Thus, Lhx4 and Prop1 have critical, but mechanistically different roles in specification and expansion of specialized anterior pituitary cells.

Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion

Development ◽  
1996 ◽  
Vol 122 (1) ◽  
pp. 151-160 ◽  
Author(s):  
P.J. Gage ◽  
M.L. Roller ◽  
T.L. Saunders ◽  
L.M. Scarlett ◽  
S.A. Camper
Keyword(s):  
Anterior Pituitary ◽  
Cell Lineage ◽  
Cell Types ◽  
Limited Commitment ◽  
Recessive Mutation ◽  
Pituitary Cells ◽  
Lineage Specification ◽  
Ames Dwarf ◽  
Dependent Cell ◽  
A Cell

The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cells lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters containing different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df<-->+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.

scholarly journals SAT-298 Integrative Single-Cell Transcriptomic and Epigenomic Landscape of Mouse Anterior Pituitary Cell Types

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Frederique Murielle Ruf-Zamojski ◽  
Michel A Zamojski ◽  
German Nudelman ◽  
Yongchao Ge ◽  
Natalia Mendelev ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Pituitary Cell ◽  
Integrated Analysis ◽  
Pituitary Cells ◽  
Rna Seq ◽  
Cell Type

Abstract The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity. Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

Cell-density dependence of the rate of prolactin secretion from perifused rat anterior pituitary cells

1983 ◽  
Vol 97 (2) ◽  
pp. 221-228 ◽  
Author(s):  
A. M. Bentley ◽  
M. Wallis
Keyword(s):  
Density Dependence ◽  
Cell Density ◽  
Anterior Pituitary ◽  
Prolactin Secretion ◽  
Female Rats ◽  
Pituitary Cells ◽  
The Third ◽  
Pituitary Glands ◽  
Prolactin Content ◽  
Perifusion System

Anterior pituitary glands from female rats were dispersed enzymically in the absence of dopamine. Dispersed cells (106–107) were layered onto columns containing Bio-Gel P-2 and were then perifused for 3 h with Dulbecco's Modified Eagle's Medium. The prolactin content of the perifusate and cell homogenates was determined by radioimmunoassay. Prolactin secretion during the third hour of perifusion increased as the loading of cells increased. However, the increase was not linear, and when secretion rate per 106 cells was calculated it was found that increased loading decreased the rate, which fell to a plateau of 1·3 ± 0·1 (s.e.m.) ng/min per 106 cells at a loading of about 8 × 106 cells from 3·8 ± 0·1 ng/min per 106 cells for a loading of 106 cells. This cell-density dependence of the rate of prolactin secretion in the perifusion system may be due to intercellular contact since the isolation of the tissue removes the influence of hypothalamic factors, while localized build up of prolactin (potentially causing direct autoregulation on the lactotroph) seems unlikely because of the continuous flow of medium.

scholarly journals Extracellular Localisation of the C-Terminus of DDX4 Confirmed by Immunocytochemistry and Fluorescence-Activated Cell Sorting

Cells ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 578 ◽  
Author(s):  
Yvonne L. Clarkson ◽  
Emma Weatherall ◽  
Martin Waterfall ◽  
Marie McLaughlin ◽  
Haojiang Lu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Sorting ◽  
Ovarian Tissue ◽  
Fluorescence Activated Cell Sorting ◽  
Rt Pcr ◽  
Rna Helicases ◽  
Dead Box ◽  
C Terminus ◽  
Expression Construct ◽  
C And N

Putative oogonial stem cells (OSCs) have been isolated by fluorescence-activated cell sorting (FACS) from adult human ovarian tissue using an antibody against DEAD-box helicase 4 (DDX4). DDX4 has been reported to be germ cell specific within the gonads and localised intracellularly. White et al. (2012) hypothesised that the C-terminus of DDX4 is localised on the surface of putative OSCs but is internalised during the process of oogenesis. This hypothesis is controversial since it is assumed that RNA helicases function intracellularly with no extracellular expression. To determine whether the C-terminus of DDX4 could be expressed on the cell surface, we generated a novel expression construct to express full-length DDX4 as a DsRed2 fusion protein with unique C- and N-terminal epitope tags. DDX4 and the C-terminal myc tag were detected at the cell surface by immunocytochemistry and FACS of non-permeabilised human embryonic kidney HEK 293T cells transfected with the DDX4 construct. DDX4 mRNA expression was detected in the DDX4-positive sorted cells by RT-PCR. This study clearly demonstrates that the C-terminus of DDX4 can be expressed on the cell surface despite its lack of a conventional membrane-targeting or secretory sequence. These results validate the use of antibody-based FACS to isolate DDX4-positive putative OSCs.

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