Progenitor cells in the embryonic anterior pituitary abruptly and concurrently depress mitotic rate before progressing to terminal differentiation

The control of progenitor cell proliferation in concert with terminal differentiation during embryonic development is poorly understood. The present paper examines this issue in the different cell lineages of the fetal mouse pituitary. Mouse fetuses were pulse-exposed to 3H-thymidine (3H-T) on a single day between embryonic day (E) 10 and E16 (prior to the onset of hormone phenotype expression) and the 3H-T labeling index of each cell type determined 3 or 4 days later (E13-19), when hormone phenotypes were detectable. In the pars tuberalis primordium, TSHbeta appeared from E13. Of these cells 75.5% were labeled when 3H-T had been administered on E10. Label decreased to 40.8% when it had been incorporated on E11 and was negligible (4.2%) when it had been taken up on E12. In the pars distalis, ACTH appeared on E13, TSHbeta, and PRL on E14, LHbeta/FSHbeta on E15 and GH on E16. When examined on E16, all these cell types were labeled for 50-60% if 3H-T had been injected on E12, but this number dropped to about 15% when 3H-T had been given on E13. Only 5-10% of the hormonal cells had taken up label when E14, 15, and 16 were the days of 3H-T administration. The decline in overall labeling index (LI) within both parts of the pituitary was significantly smaller than that in the hormone expressing cells. It is concluded that an outspoken decline in proliferation of the cells destined to become hormone-expressing cell types occurs one to several days before these hormones come to expression. In the pars distalis, this decline occurs at a common time point i.e. between E12 and E13 for each cell type. Pars tuberalis and pars distalis TSHbeta cells show distinct 3H-T labeling profiles, suggesting distinct cell lineage sources for each.status: publishe

Triiodothyronine expands the lactotroph and maintains the lactosomatotroph population, whereas thyrotrophin-releasing hormone augments thyrotroph abundance in aggregate cell cultures of postnatal rat pituitary gland

In the present study, we used a three-dimensional pituitary cell culture system from early postnatal rats to examine the in vitro developmental potential of triiodothyronine (T3) and thyrotrophin-releasing hormone (TRH). Cell types were identified at the hormone mRNA level by single-cell reverse transcription-polymerase chain reaction and any change in abundance was further examined by immunofluorescence staining of the corresponding hormone protein. In aggregates from 14-day-old rats, long-term (12-16 days) treatment with T3 (0.5 nM) increased the abundance of cells expressing prolactin mRNA (PRLmRNA cells) by 2.5-fold and lowered that of nonhormonal cells and thyroid-stimulating hormone beta (TSHbeta)mRNA cells. The abundance of growth hormone (GH)mRNA cells decreased during culture compared to that in the freshly dispersed pituitary gland and T3 did not significantly affect this cell population. Cells coexpressing PRL mRNA and GH mRNA virtually disappeared during culture but reappeared in the presence of T3. T3 increased the abundance of PRL-immunoreactive (ir) cells in aggregates from 14-day-old rats, as well as in aggregates from newborn and 1-week-old rats. As estimated by bromodeoxyuridine (BrdU) labelling, a 3-day treatment with T3 enhanced the number of PRL-ir cells that had incorporated BrdU, but did not yet expand the total population of PRL-ir cells. Long-term treatment with TRH (100 nM) did not affect the proportion of PRLmRNA or GHmRNA cells, but consistently increased the proportional number of TSHbeta(mRNA) and TSHbeta-ir cells. The present data confirm the findings obtained in recent in vivo loss of function genetic studies suggesting that T3 plays a prominent role in postnatal expansion of the lactotroph population and that TRH is important for thyrotroph development. The data suggest that the effect of T3 is brought about by a direct action on the pituitary gland through a cell proliferation mechanism. T3 also appears to support the lactosomatotroph population. In view of the established theory that lactotrophs develop from GH-expressing progenitor cells and that this is a post mitotic event, we propose that T3 is mitogenic for GHmRNA cells that lack GH-ir material and that transdifferentiate into PRL-ir cells, but that a pathway of PRL cell development from mitotic nonhormonal cell progenitors may also be involved.status: publishe

Stimulation of combinatorial expression of prolactin and glycoprotein hormone alpha-subunit genes by gonadotropin-releasing hormone and estradiol-17beta in single rat pituitary cells during aggregate cell culture

Previously we showed the existence of rat and mouse anterior pituitary cells coexpressing mRNA from two or more hormone genes in which production and/or storage of the corresponding hormones were not detectable. To substantiate a putative function for these cells, we investigated whether these phenotypes were retained during long-term reaggregate cell culture and whether protagonist regulatory factors could expand cell populations expressing particular hormone mRNA combinations. After 4-wk culture and treatments, aggregates were trypsinized and single cells collected by means of a fluo-rescence-activated cell sorter. Hormone mRNAs were detected by single-cell RT-PCR. Combinatorial hormone mRNA expression was retained in culture. Both estradiol (E2) and GnRH (1 nM) markedly augmented the proportion of cells expressing prolactin (PRL) mRNA together with other hormone mRNAs and cells expressing glycoprotein subunit (GSU)-alpha mRNA together with other hormone mRNAs. GnRH strongly increased the proportion of cells containing alphaGSU mRNA alone, but E2 did not. GnRH and (E2) affected the expansion of a population (approximately 20% of all cells) coexpressing PRL and alphaGSU mRNA without betaGSUs. Immunostaining of stored hormone on tissue sections revealed colocalization of PRL and alphaGSU in the E2- but not in the GnRH-treated cells. The present findings suggest that cells coexpressing different pituitary hormone mRNAs form a distinct population that survives without extrapituitary factors. Their occurrence can be markedly modified by regulatory factors. Certain hormone regimens favor unique coexpressions distinctly at mRNA and protein level. These peculiar characteristics support the notion that combinatorial expression of hormone genes in the pituitary serves a biological role.status: publishe

Ontogeny of plurihormonal cells in the anterior pituitary of the mouse, as studied by means of hormone mRNA detection in single cells

The expression of mRNA of growth hormone (GH), prolactin (PRL), pro-opiomelanocortin (POMC) and the common glycoprotein hormone alpha-subunit (alphaGSU) was studied by means of single cell reverse transcriptase-polymerase chain reaction in male mouse pituitary cells at key time points of fetal and postnatal development: embryonic day 16 (E16); postnatal day 1 (P1) and young-adult age (P38). At E16, the hormone mRNAs examined were detectable, although only in 44% of total cells. Most of the hormone-positive cells expressed only one of the tested hormone mRNAs (monohormonal) but 14% of them contained more than one hormone mRNA (plurihormonal cells). Combinations of GH mRNA with PRL mRNA, of alphaGSU mRNA with GH and/or PRL mRNA and of POMC mRNA with GH and/or PRL mRNA or alphaGSU mRNA were found. As expected, the proportion of hormone-positive cells rose as the mouse aged. The proportions of plurihormonal cells followed a developmental pattern independent of that of monohormonal cells and characteristic for each hormone mRNA examined. Cells coexpressing POMC mRNA with GH or PRL mRNA significantly rose in proportion between E16 and P1, while the proportion of cells coexpressing GH and PRL mRNA markedly increased between P1 and P38. The occurrence of cells displaying combined expression of alphaGSU mRNA with GH and/or PRL mRNA did not significantly change during development. Remarkably, the population of cells expressing PRL mRNA only, was larger at E16 than at P1 and expanded again thereafter. In conclusion, the normal mouse pituitary develops a cell population that is capable of expressing multiple hormone mRNAs, thereby combining typical phenotypes of different cell lineages. These plurihormonal cells are already present during embryonic life. This population is of potential physiological relevance because development-related factors appear to determine which hormone mRNAs are preferentially coexpressed. Coexpression of multiple hormone mRNAs may represent a mechanism to respond to temporally increased endocrine demands. The data also suggest that the control of combined hormone expression is different from that of single hormone expression, raising questions about the current view on pituitary cell lineage specifications.status: publishe

Presence of gonadotropin-releasing hormone (GnRH) mRNA in Rathke's pouch and effect of the GnRH-antagonist ORG 30276 on lactotroph development in vitro

Reverse transcription-polymerase chain reaction (RT-PCR) with specific GnRH cDNA primers performed on RNA from Rathke's pouches removed from pregnant rats at day 12 of gestation (e12) generated an amplified DNA fragment of the expected length (357 bp). The fragment hybridized with a labeled GnRH cDNA probe in Southern blotting. DNA sequencing demonstrated identity with the known nucleotide sequence of the corresponding segment of rat GnRH cDNA. To determine whether GnRH mRNA was located in the Rathke's pouch cells or in remnants of surrounding tissue not completely removed during preparation, the pouches were treated with collagenase. Based on light and electron microscopic examination, this treatment disconnected virtually all contaminating tissue, allowing the 'pure' Rathke's pouches to be picked-up separately. Again, RT-PCR generated a DNA fragment of the expected length, the fragment hybridized with the GnRH cDNA probe and showed the nucleotide sequence of the corresponding region of rat GnRH cDNA. In Rathke's pouches established in explant culture on e12, lactotrophs were well developed when examined 9 days later by immunostaining of prolactin in paraffin-embedded sections of the tissue. Computerized image analysis showed prolactin immunoreactivity in 8.0+/-1.1% of the section area. Addition of the potent and long-acting GnRH antagonist ORG 30276 to the crude preparation of Rathke's pouches caused a significant decrease in the relative area staining for prolactin. The latter effect was abolished by concomitant addition of GnRH. In preparations of pure Rathke's pouches (collagenase-treated), ORG 30276 failed to affect the relative area of prolactin immunoreactivity. GnRH mRNA remained expressed in explants of both crude and pure Rathke's pouches until the end of the culture period. It is concluded that the GnRH gene is expressed in Rathke's pouch as early as e12 and that GnRH may be a physiological paracrine/autocrine peptide stimulating the development of lactotrophs. Mesenchymal and/or neural factors may be essential for the latter system to function.status: publishe

Nestin-immunoreactive cells in rat pituitary are neither hormonal nor typical folliculo-stellate cells

Nestin is an intermediate filament protein that has originally been identified as a marker of neuroepithelial stem/progenitor cells. The present study explored whether nestin immunoreactivity (nestin-ir) is present in the rat pituitary and in which cell type(s). Nestin-ir was observed in scattered cells in the anterior, intermediate, and neural lobes. Nestin-ir cells were predominantly of stellate shape and were more numerous in immature than in adult animals. Nestin-ir did not colocalize with any pituitary hormone, and did not colocalize or only very sporadically with the folliculo-stellate cell marker S100. In the intermediate lobe, nestin-ir cells contained glial fibrillary acidic protein in an age-dependent manner. Nestin-ir cells were closely associated with endothelial and fibronectin-ir cells, but did mostly not coincide. Nestin-ir was not found in alpha-smooth muscle actin-ir myofibroblasts or in microglial cells. Regardless of age, nestin-ir was detected in some unidentifiable cells that border the pituitary cleft. Nestin-ir remained present in pituitary cultured as three-dimensional aggregates. Treatment with basic fibroblast growth factor or leukemia inhibitory factor increased the number of nestin-ir cells. Starting from anterior lobe cell monolayer cultures, nestin-ir cells could be selected and propagated to a virtually pure population. These nestin-ir cells displayed remarkable motility and proliferative activity, and did not express hormones, glial fibrillary acidic protein, or S100, but contained vimentin-, fibronectin-, and alpha-smooth muscle actin-ir. In conclusion, nestin-ir is present in the pituitary in cells that are neither hormonal nor typical folliculo-stellate. The expression pattern depends on age and lobe examined. Pericapillar localization suggests a pericyte phenotype for some of them. Whether the heterogeneous nestin-ir population also contains pituitary progenitor cells remains to be explored.status: publishe

Combinatorial expression of phenotypes of different cell lineages in the rat and mouse pituitary

As studied by single cell RT-PCR of pituitary hormones, we demonstrated that the pituitaries of rats and mice contain a subpopulation of cells that express two or more hormone phenotypes typically belonging to lineages that are branched separately early during embryonic development, such as glycoprotein hormone alpha-subunit (alphaGSU) mRNA + PRL mRNA, alphaGSU mRNA + POMC mRNA, and POMC mRNA + GH or PRL mRNA. GnRH in vitro selectively expands the population of cells coexpressing alphaGSU mRNA + PRL mRNA, and CRH selectively increases the proportion of cells coexpressing alphaGSU mRNA + POMC mRNA. Colocalization of alphaGSU + PRL or alphaGSU + POMC could not be detected by double immunofluorescence. This lineage promiscuity was also observed in the pituitary in vivo.status: publishe