Roles of differential expression of miR-543-5p in GH regulation in rat anterior pituitary cells and GH3 cells.

Growth hormone (GH) is an important hormone released by the pituitary gland that plays a key role in the growth and development of organisms. In our study, TargetScan analysis and the dual luciferase reporter assays were used to predict and screen for miRNAs that might act on the rat Gh1 gene, and we identified miR-543-5p. Then, the GH3 cell line and the primary rat pituitary cells were transfected with miRNA mimic, inhibitor, and siRNA. We detected the Gh1 gene expression and the GH secretion by real-time PCR and ELISAs, respectively, to verify the regulatory effect of miR-543-5p on GH secretion. The results showed that miR-543-5p can inhibit Gh1 mRNA expression and reduce GH secretion. MiR-543-5p inhibitor upregulated Gh1 mRNA expression and increased GH secretion compared with the negative control. In summary, miR-543-5p downregulates Gh1 expression, resulting in a decrease in GH synthesis and secretion, which demonstrates the important role of miRNAs in regulating GH and animal growth and development.

Disruption of the Pituitary Circadian Clock Induced by Hypothyroidism and Hyperthyroidism: Consequences on Daily Pituitary Hormone Expression Profiles.

BACKGROUND: The secretion of pituitary hormones oscillates throughout the 24-hour period, indicating that circadian clock-mediated mechanisms regulate this process in the gland. Additionally, pituitary hormone synthesis has been shown to be altered in hypo- and hyperthyroidism. Although thyroid hormones can modulate the other peripheral clocks, the interaction between thyroid hormone levels and circadian clock gene expression in the anterior pituitary has yet to be elucidated. METHODS: Male Wistar rats were divided into three groups: control, hypothyroid, and hyperthyroid. Following the experimental procedures, animals were euthanized every three hours over the course of a 24-hour period. The anterior pituitary glands were excised and processed for mRNA expression analysis by quantitative reverse transcriptase polymerase chain reaction. One- and two-way analysis of variance as well as cosinor analysis were used to evaluate the time-of-day-dependent differential expression for each gene in each experimental group and their interactions. RESULTS: Hyperthyroidism increased the mRNA expression of core clock genes and thyrotrophic embryonic factor (Tef), as well as the mesor and amplitude of brain and muscle Arnt-like protein-1 (Bmal1) and the mesor of nuclear receptor subfamily 1 (Nr1d1) group D member 1, when compared to euthyroid animals. Hypothyroidism disrupted the circadian expression pattern of Bmal1 and period circadian regulator 2 (Per2) and decreased the mesor of Nr1d1 and Tef. Furthermore, it was observed that the pituitary content of Dio2 mRNA was unaltered in hyperthyroidism but substantially elevated in hypothyroidism during the light phase. The upregulated expression was associated with an increased mesor and amplitude, along with an advanced acrophase. The gene expression of all the pituitary hormones was found to be altered in hypo- and hyperthyroidism. Moreover, prolactin (Prl) and luteinizing hormone beta subunit (Lhb) displayed circadian expression patterns in the control group, which were disrupted in both the hypo- and hyperthyroid states. CONCLUSION: Taken together, the data demonstrate that hypo- and hyperthyroidism alter circadian clock gene expression in the anterior pituitary. This suggests that triiodothyronine plays an important role in the regulation of pituitary gland homeostasis, which could ultimately influence the rhythmic synthesis and/or secretion of all the anterior pituitary hormones.

FOXA2 gene mutation in a patient with congenital complex pituitary hormone deficiency.

We report a patient with congenital complex pituitary hormone deficiency (CPHD) with intestinal malrotation and anal atresia. We identified a de novo heterozygous mutation, c.664T > G (p.Cys222Gly), in the FOXA2 gene in this individual. This missense mutation had the potential to affect the DNA binding properties of the FOXA2 protein based on a protein structure prediction. Since a CPHD patient with another missense mutation and one other case with an entire gene deletion have also been reported, we speculated that a haploinsufficiency of the FOXA2 gene might be a genetic etiology for this disorder. Phenotypic similarities and differences among these three cases are also discussed.

Induction of chemokines and prostaglandin synthesis pathways in luteinized human granulosa cells: potential role of luteotropin withdrawal and prostaglandin F2α in regression of the human corpus luteum.

Our objective was to determine the effects of prostaglandin F2α (PGF2α) and withdrawal of luteotropic stimulants (forskolin or hCG) on expression of chemokines and prostaglandin-endoperoxide synthase 2 (PTGS2) in luteinized human granulosa cells. Human granulosa cells were collected from 12 women undergoing oocyte retrieval and were luteinized in vitro with forskolin or hCG. In first experiment, granulosa-lutein cells were treated with PGF2α, the primary luteolytic hormone in most species. In second experiment, granulosa cells that had been luteinized for 8 d had luteotropins withdrawn for 1, 2, or 3 d. Treatment with PGF2α induced mRNA for chemokine (c-x-c motif) ligand 2 (CXCL2) and CXC ligand 8 (CXCL8; also known as interleukin-8) in granulosa cells luteinized for 8 d but not in cells that were only luteinized for 2 d. Similarly, luteinization of human granulosa cells for 8 d with forskolin or hCG followed by withdrawal of luteotropic stimulants, not only decreased P4 production, but also increased mRNA concentrations for CXCL8, CXCL-2 (after forskolin withdrawal), and PTGS2. These results provide evidence for two key steps in differentiation of luteolytic capability in human granulosa cells. During 8 d of luteinization, granulosa cells acquire the ability to respond to luteolytic factors, such as PGF2α, with induction of genes involved in immune function and PG synthesis. Finally, a decline in luteotropic stimuli triggers similar pathways leading to induction of PTGS2 and possibly intraluteal PGF2α production, chemokine expression, leukocyte infiltration and activation, and ultimately luteal regression.

Circulating microRNA profiles and the identification of miR-593 and miR-511 which directly target the PROP1 gene in children with combined pituitary hormone deficiency.

Since the tissue of children with combined pituitary hormone deficiency (CPHD) is not readily accessible, a new focus in children with CPHD is the blood-based expression profiling of non-protein coding genes, such as microRNAs (miRNAs or miRs), which regulate gene expression by inhibiting the translation of mRNAs. In this study, to address this, we identified potential miRNA signatures for CPHD by comparing genome-wide miRNA expression profiles in the serum of children with CPHD vs. normal (healthy) controls. Human embryonic kidney 293T cells were transfected with miR-593 or miR-511 oligonucleotides. Potential target gene expression was validated by western blot analysis for proteins and by miR-593 or miR-511 reporter assay using PROP1 gene 3'-untranslated region (3'-UTR) reporter. The miR-593 and miR-511 levels in the serum of 103 children with CPHD were assessed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. We found 23 upregulated and 19 downregulated miRNAs with abnormal expression in children with CPHD compared with the normal controls using miRNA microarray analysis and RT-qPCR. miR-593 and miR-511 targeted the 3'-UTR of the PROP1 gene and attenuated the expression of PROP1. The levels of miR-593 and miR-511 in the serum of children with CPHD were increased compared with those in the control subjects. According to Youden's index, the sensitivity was 82.54 and 84.86%, and the specificity was 98.15 and 91.36% for miR-593 and miR-511, respectively. The various levels of specific miRNAs, particularly miR-593 and miR-511 whose direct target is the PROP1 gene, may serve as a non-invasive diagnostic biomarkers for children with CPHD.

Dioxin-induced retardation of development through a reduction in the expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: a comparative study using two strains of mice with different sensitivities to dioxin.

We have previously revealed that treating pregnant rats with 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) reduces the expression of gonadotropins and growth hormone (GH) in the fetal and neonatal pituitary. A change in gonadotropin expression impairs the testicular expression of steroidogenic proteins in perinatal pups, and imprint defects in sexual behavior after reaching maturity. In this study, we examined whether TCDD also affects the expression of gonadotropin and GH in mice using C57BL/6J and DBA/2J strains which express the aryl hydrocarbon receptor (Ahr) exhibiting a different affinity for TCDD. When pregnant C57BL/6J mice at gestational day (GD) 12 were given oral TCDD (0.2-20 µg/kg), all doses significantly attenuated the pituitary expression of gonadotropin mRNAs in fetuses at GD18. On the other hand, in DBA/2J mice, a much higher dose of TCDD (20 µg/kg) was needed to produce a significant attenuation. Such reduction in the C57BL/6J strain continued until at least postnatal day (PND) 4. In agreement with this, TCDD reduced the testicular expression of steroidogenic proteins in C57BL/6J neonates at PND2 and 4, although the same did not occur in the fetal testis and ovary. Furthermore, TCDD reduced the perinatal expression of GH, litter size and the body weight of newborn pups only in the C57BL/6J strain. These results suggest that 1) also in mice, maternal exposure to TCDD attenuates gonadotropin-regulated steroidogenesis and GH expression leading to the impairment of pup development and sexual immaturity; and 2) Ahr activation during the late fetal and early postnatal stages is required for these defects.

Phosphatase and tensin homologue and pituitary tumor-transforming gene in pituitary adenomas. Clinical-pathologic and immunohistochemical analysis.

Pituitary tumor-transforming gene (PTTG) is also known to induce angiogenesis during pituitary tumorigenesis. It has not been clarified whether PTTG functions as a cytoplasmic or a nuclear protein. Pituitary tumor-transforming gene-1 is usually expressed in most pituitary tumors, and little is known about phosphatase and tensin homologue (PTEN). In our knowledge, it has not been studied in pituitary tumors. The aim of this study was to determine the correlation between proliferating cell nuclear antigen (PCNA) labeling index (LI), PTEN, and PTTG-1 immunoexpression in pituitary adenomas. Forty-five pituitary adenomas were included-46.7% were males and 53.7% were females. The mean age was 43.18 +/- 9.42 years (27-70 years). For functional pituitary adenoma (PA), it was 41.92 +/- 6.63, and for nonfunctional pituitary adenomas, it was 44.62 +/- 11.85 (P = .003). Proliferating cell nuclear antigen LI range was 19.42 +/- 5.49; in functional pituitary adenomas, it was 41.92 +/- 6.63, and in nonfunctional adenomas, it was 44.62 +/- 11.85 (P = .081). The PTEN immunoreaction was positive-weak in 21 (47%), moderate in 19 (42%), and strong in 5 (11%; P = .000). The PTTG-1 gene was positive-weak in 18 (41%), moderate in 19 (41%), and strong in 6 (13%; P = .000). When we correlated PTEN + PCNA, it was P =.004, and PTEN + PTTG-1, it was P = .019. And when we correlated PCNA + PTGG-1, it was P = .262. In our results, we observed higher expression of PCNA-LI and PTTG-1 and loss of expression of PTEN. Nonfunctional hypophysis adenomas presented a higher PCNA, PTTG-1, and PTEN expression than functional ones. There was no difference between single-hormone-producing hypophysis adenomas or multiple-hormone-producing ones. Necrosis and hemorrhage were associated with PTEN expression, whereas atypias and mitosis figures were associated to PTTG-1 expression.

Retention of genes involved in the adenohypophysis-mediated endocrine system in early vertebrates.

The adenohypophysis of vertebrates receives peptide hormones from the hypothalamus and secretes hormones that regulate diverse physiologic processes in peripheral organs. The adenohypophysis-mediated endocrine system is widely conserved across vertebrates but not invertebrates. Phylogenetic analysis indicates that the emergence of this system coincided with two rounds of whole-genome duplication (2R-WGD) in early vertebrates, but direct evidence linking these events has been unavailable. We detected all human paralogons (series of paralogous regions) formed in early vertebrates as traces of 2R-WGD, and examined the relationship between 2R-WGD and the evolution of genes essential to the adenohypophysis-mediated endocrine system. Regarding genes encoding transcription factors (TFs) involved in the terminal differentiation into hormone-secreting cells in adenohypophyseal development, we showed that most pairs of these genes and their paralogs were part of paralogons. In addition, our analysis also indicated that most of the paralog pairs in families of adenohypophyseal hormones and their receptors were part of paralogons. These results suggest that 2R-WGD played an important role in generating genes encoding adenohypophyseal TFs, hormones, and their receptors for increasing the diversification of hormone repertoire in the adenohypophysis-mediated endocrine system of vertebrates.

The dawn and evolution of hormones in the adenohypophysis.

The adenohypophysial hormones have been believed to have evolved from several ancestral genes by duplication followed by evolutionary divergence. To understand the origin and evolution of the endocrine systems in vertebrates, we have characterized adenohypophysial hormones in an agnathan, the sea lamprey Petromyzon marinus. In gnathostomes, adrenocorticotropin (ACTH) and melanotropin (MSH) together with beta-endorphins (beta-END) are encoded in a single gene, designated as proopiomelanocortin (POMC), however in sea lamprey, ACTH and MSH are encoded in two distinct genes, proopoicortin (POC) gene and proopiomelanotropin (POM) gene, respectively. The POC and POM genes are expressed specifically in the rostral pars distalis (RPD) and the pars intermedia (PI), respectively. Consequently, the final products from both tissues are the same in all vertebrates, i.e., ACTH from the PD and MSH from the PI. The POMC gene might have been established in the early stages of invertebrate evolution by internal gene duplication of the MSH domains. The ancestral gene might be then inherited in lobe-finned fish and tetrapods, while internal duplication and deletion of MSH domains as well as duplication of whole POMC gene took place in lamprey and gnathostome fish. Sea lamprey growth hormone (GH) is expressed in the cells of the dorsal half of the proximal pars distalis (PPD) and stimulates the expression of an insulin-like growth factor (IGF) gene in the liver as in other vertebrates. Its gene consists of 5 exons and 4 introns spanning 13.6 kb, which is the largest gene among known GH genes. GH appears to be the only member of the GH family in the sea lamprey, which suggests that GH is the ancestral hormone of the GH family that originated first in the molecular evolution of the GH family in vertebrates and later, probably during the early evolution of gnathostomes. The other member of the gene family, PRL and SL, appeared by gene duplication. A beta-chain cDNA belonging to the gonadotropin (GTH) and thyrotropin (TSH) family was cloned. It is expressed in cells of the ventral half of PPD. Since the expression of this gene is stimulated by lamprey gonadotropin-releasing hormone, it was assigned to be a GTHbeta. This GTHbeta is far removed from beta-subunits of LH, FSH, and TSH in an unrooted tree derived from phylogenetic analysis, and takes a position as an out group, suggesting that lampreys have a single GTH gene, which duplicated after the agnathans and prior to the evolution of gnathostomes to give rise to LH and FSH.

Transcriptional control during mammalian anterior pituitary development.

The mammalian anterior pituitary gland is a compound endocrine organ that regulates reproductive development and fitness, growth, metabolic homeostasis, the response to stress, and lactation, by actions on target organs such as the gonads, the liver, the thyroid, the adrenals, and the mammary gland. The protein and peptide hormones that control these physiological parameters are secreted by specialized pituitary cell types that derive from a common origin in the early ectoderm. Collectively, the broad physiological importance of the pituitary gland, its intriguing organogenesis, and the clinical and agricultural significance of its actions, have established pituitary development as an excellent model system for the study of the gene-regulatory cascades that guide vertebrate cell determination and differentiation. We review the transcriptional pathways that regulate the commitment of the individual pituitary cell lineages and that subsequently modulate trophic hormone gene activity in the differentiated cells of the mature gland.

Current approaches for deciphering the molecular basis of combined anterior pituitary hormone deficiency in humans.

This review focuses on the general strategies currently used to decipher the molecular bases of combined pituitary hormone deficiency (CPHD) of genetic origin. By summarizing illustrative approaches that turned out to be successful for identifying an increasing number of genes involved in CPHD in the human, this article consider predictable obstacles specific to the investigation of these rare and heterogeneous conditions, while underlining the previously unsuspected roles of several of these genes during the development of extrapituitary structures.

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.

The Pars tuberalis of the monkey (Macaca fascicularis) hypophysis: cell types and hormone expression.

The pars tuberalis (Pt) of most mammalian species contains specific cells which are structurally and functionally different from the pars distalis (Pd) cells. Pt-specific cells possess melatonin receptors and reveal morphological changes dependent on the duration of the photoperiod. Furthermore, in hamsters the transmission of photoperiodic stimuli to the endocrine system is influenced by melatonin, an effect which is likely to be mediated by Pt-specific cells. In monkeys, however, only little is known about this cell type. Therefore, we studied the ultrastructural differentiation of Pt-specific cells and describe the expression of different hormones and their mRNA by immunohistochemistry and in situ hybridization. Apparently the Pt consists of (1) cells similar to gonadotropic cells of the Pd, (2) folliculostellate cells and (3) a cell population which is morphologically and functionally clearly distinct from all other cell types found in the Pd. Morphologically they resemble the Pt-specific cells found in other species. Regarding the expression of secretory products there is evidence that they transcribe and translate the beta-TSH subunit. Although there is a strong signal for the mRNA of the common alpha-chain, protein staining is much weaker. POMC mRNA is expressed in the Pt while there is no evidence for PRL mRNA. The present results lead to the conclusion that the Pt of the monkey contains Pt-specific cells which express different hormonal subunits as was already shown for other species. In context with previous findings of melatonin receptors in the monkey Pt further investigations are necessary to establish the possible role of Pt-specific cells in the photoperiod-dependent generation of endocrine rhythms.

Construction and in vivo efficacy of a replication-deficient recombinant adenovirus encoding murine growth hormone.

We have constructed a recombinant, replication-deficient, first-generation adenovirus-encoding mouse GH (mGH), AdCMVmGH. This virus directed mGH production from an epithelial cell line in vitro in a dose-dependent manner. When injected into the quadriceps muscle or submandibular ducts of mGH-deficient Snell dwarf mice, AdCMVmGH resulted in the production of significantly elevated serum mGH levels. Furthermore, after i.m. injection, dwarf mice increased in weight by 8% over 4 days and close to 100% by 30 days. When AdCMVmGH was administered to 3- to 4-week-old rats by i.v. injection to assess general metabolic responses, serum mGH, insulin-like growth factor 1, triglycerides and cholesterol levels were significantly elevated. AdCMVmGH should be a valuable experimental tool for the controlled, directed expression of mGH in preclinical mouse model studies.

Cell and molecular biology of the pars tuberalis of the pituitary.

The pars tuberalis of the adenohypophysis is mainly composed of a special type of endocrine cells, pars tuberalis-specific cells, lining the primary capillary plexus of the hypophysial portal system. Dense expression of melatonin receptors and marked changes in morphological appearance, production pattern, and secretory activity during annual cycle show that these cells are highly sensitive to changes in photoperiod. This leads to the hypothesis that the pars tuberalis is involved in the transmission of photoperiodic stimuli to endocrine targets. Several investigations support the theory that pars tuberalis-specific cells are multipotential cells exerting a modulatory influence on the secretory activity of the pars distalis. Specifically, there is accumulating evidence that seasonal modulation of prolactin secretion, independent of hypothalamic input, is due to melatonin-regulated activity of pars tuberalis-specific cells. The exact nature of secretory products and their effects within neuroendocrine regulation, however, remain rather enigmatic. Accordingly, molecular mechanisms regulating gene expression under the influence of photoperiod, respectively, circulating melatonin levels are still incomplete. Recent cloning of melatonin receptor genes and new data on intracellular signal transduction will probably lead to new insights on melatonin action and pars tuberalis-specific cell physiology.

GH transcription factors.

The pituitary transcription factor Pit-1 is expressed during the later differentiation stages of anterior pituitary development and Pit-1 mutations have been identified as the cause of a combined pituitary hormone deficiency (CPHD) for GH, prolactin and TSH. Mutations within the human Pit-1 gene can either impair the DNA binding of this transcription factor, or while leaving DNA binding capabilities unimpaired, decrease its function within the transactivation complex. Approximately half of all patients with this phenotype do not show any defect within the Pit-1 gene. Prop-1, a recently discovered transcription factor of anterior pituitary development, seemed a likely candidate for such mutations. Prop-1 mutations, however, have been found so far to induce a combined pituitary hormone deficiency for GH, prolactin, TSH and gonadotropins. We describe here a group of patients with isolated and combined pituitary hormone deficiencies who were screened for Pit-1 and Prop-1 mutations to characterize the phenotypic spectrum of defects within these two genes.

Food restriction differentially affects mRNAs encoding the major anterior pituitary tropic hormones.

Chronic food restriction (FR) leads to adaptive cellular changes, some of which retard aging. Moreover, some of these changes occur within weeks after onset of FR. Because neuroendocrine mechanisms may mediate these effects, we measured the effect of FR on the messenger ribonucleicacids (mRNAs) encoding all of the tropic hormones of the anterior pituitary (AP). Slot blot and solution hybridization were conducted on AP ribonucleicacid (RNA) samples obtained at 0500 h (AM) and 1500 h (PM) from 3-month-old male Fischer 344 rats fed ad libitum (AL) or FR (60% of AL calories) since 6 weeks of age. PolyA RNA/microgram total RNA was similar in AL and FR rats, indicating that there was no overall effect of FR on mRNA levels. The level of proopiomelanocortin (POMC) mRNA was not reduced by FR when expressed per microgram of RNA or as total AP content. By contrast, the total AP content of the mRNAs encoding LH beta, FSH beta, TSH beta, GH, and PRL was markedly reduced by FR. When expressed per microgram of RNA, however, only GH (AM and PM), FSH beta (AM), TSH beta (PM), and PRL (PM) were reduced by FR. These results reveal that FR differentially affects pituitary tropic hormone mRNA levels within weeks after onset of FR, and are consistent with a role for neuroendocrine alterations in the initiation of adaptive cellular responses to FR.

Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from a set of genes expressed at specific stages of differentiation in a given cell lineage, we demonstrate that targeted oncogenesis can produce cell lines representing sequential stages of development, in essence allowing both spatial and temporal immortalization. Our strategy was based on our production of a committed but immature pituitary gonadotrope cell line by directing expression of the oncogene SV40 T antigen using a gonadotrope-specific region of the human glycoprotein hormone alpha-subunit gene in transgenic mice. These cells synthesize alpha-subunit and gonadotropin-releasing hormone (GnRH) receptor, yet are not fully differentiated in that they do not synthesize the beta-subunits of luteinizing hormone (LH) or follicle-stimulating hormone (FSH). This observation lead to the hypothesis that targeting oncogenesis with promoters that are activated earlier or later in development might immortalize cells that were more primitive or more differentiated, respectively. To test this hypothesis, we used an LHbeta promoter to immortalize a cell that represents a subsequent stage of gonadotrope differentiation (expression of alpha-subunit, GnRH receptor, and LH beta-subunit but not FSH beta-subunit). Conversely, targeting oncogenesis with a longer fragment of the human alpha-subunit gene (which is activated earlier in development) resulted in the immortalization of a progenitor cell that is more primitive, expressing only the alpha-subunit gene. Interestingly, this transgene also immortalized cells of the thyrotrope lineage that express both alpha- and beta-subunits of thyroid-stimulating hormone and the transcription factor GHF-1 (Pit-1). Thus, targeted tumorigenesis immortalizes mammalian cells at specific stages of differentiation and allows the production of a series of cultured cell lines representing sequential stages of differentiation in a given cell lineage.