POMC Cells Research Articles

PMON122 Unveiling the Kraken- An Incidentally Found Silent Corticotroph Adenoma of the Pituitary Gland

Abstract Introduction Silent corticotroph adenomas (SCA) are an unusual and rare variant of non- functioning pituitary adenomas (NFA) that do not produce biochemical hypercortisolism or clinical Cushing's disease (CD), yet stain strongly for ACTH on immunohistochemistry. They represent 3-19% of non-functioning adenomas and 20% of corticotroph producing adenomas. SCAs are distinguished by their late presentation, frequent cavernous sinus involvement and earlier recurrence requiring multimodal management. Case We present a case of a 70 year old female with a remote history of breast cancer (2002) and uterine cancer (2016) in remission who was found to have an incidental soft tissue mass in the sella turcica on CT head after a fall in 2021. An MRI brain confirmed the mass as a 1.8×2.0×1.5 cm pituitary lesion with expansion to the sella. Patient had no symptoms related to pituitary hyperfunction or mass effect. Biochemical workup including IGF-1, prolactin, LH, FSH, and AM Cortisol level were all within normal limits. Ophthalmologic evaluation was normal. Repeat pituitary MRI 2 months later showed increasing homogeneously enhancing lesion on the left petrous ridge measuring 2.8×2.8×1.3 cm. There was extension into the cavernous sinus with involvement and encasement of the carotid artery. After multi-disciplinary evaluation including neurosurgery and ENT, patient elected to undergo transnasal endoscopic resection of pituitary tumor. The tumor was only partially resected due to its close association with the carotid artery. Final pathology was significant for monotonous proliferation of tumor cells with small round nuclei and amphophilic cytoplasm, characteristic of a pituitary adenoma. The tumor cells stained strongly positive for ACTH, and negative for all other hormone receptors. Further endocrine workup showed normal morning cortisol and ACTH and marginally elevated 24 hour urine cortisol measuring 47.6 mcg/g creatinine (ULN 42.3). Her clinical presentation was consistent with a SCA. A Dotatate PET/CT was ordered to be performed 3 months postoperatively to assess for SSTR receptors for potential targeted therapy against the residual tumor. Discussion We present the case of an elderly female who was incidentally found to have an invasive SCA precluding its complete resection. There are multiple theories regarding the lack of biological activity of SCAs including lack of enzyme in the tumor to cleve the active ACTH from the prohormone state, dysfunctional lysosomal activity leading to destruction of ACTH before secretion, origin from POMC positive pars intermedia cells instead of true corticotropic cells. These tumors have an increased rate of tumor invasion and recurrence making them difficult to treat. A potential targeted treatment may be somatostatin receptor ligands which will be seen on the dotatate PET/CT. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The co-chaperone FKBP51 modulates HPA axis activity and age-related maladaptation of the stress system in pituitary proopiomelanocortin cells

Glucocorticoid (GC)-mediated negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis, the body’s physiological stress response system, is tightly regulated and essential for appropriate termination of this hormonal cascade. Disturbed regulation and maladaptive response of this axis are fundamental components of multiple stress-induced psychiatric and metabolic diseases and aging. The co-chaperone FK506 binding protein 51 (FKBP51) is a negative regulator of the GC receptor (GR), is highly stress responsive, and its polymorphisms have been repeatedly associated with stress-related disorders and dysfunctions in humans and rodents. Proopiomelanocortin (Pomc)-expressing corticotropes in the anterior pituitary gland are one of the key cell populations of this closed-loop GC-dependent negative feedback regulation of the HPA axis in the periphery. However, the cell type-specific role of FKBP51 in anterior pituitary corticotrope POMC cells and its impact on age-related HPA axis disturbances are yet to be elucidated. Here, using a combination of endogenous knockout and viral rescue, we show that male mice lacking FKBP51 in Pomc-expressing cells exhibit enhanced GR-mediated negative feedback and are protected from age-related disruption of their diurnal corticosterone (CORT) rhythm. Our study highlights the complexity of tissue- and cell type-specific, but also cross-tissue effects of FKBP51 in the rodent stress response at different ages and extends our understanding of potential targets for pharmacological intervention in stress- and age-related disorders.

POMC neuronal heterogeneity in energy balance and beyond: an integrated view.

Hypothalamic AgRP and POMC neurons are conventionally viewed as the yin and yang of the body's energy status, since they act in an opposite manner to modulate appetite and systemic energy metabolism. However, although AgRP neurons' functions are comparatively well understood, a unifying theory of how POMC neuronal cells operate has remained elusive, probably due to their high level of heterogeneity, which suggests that their physiological roles might be more complex than initially thought. In this Perspective, we propose a conceptual framework that integrates POMC neuronal heterogeneity with appetite regulation, whole-body metabolic physiology and the development of obesity. We highlight emerging evidence indicating that POMC neurons respond to distinct combinations of interoceptive signals and food-related cues to fine-tune divergent metabolic pathways and behaviours necessary for survival. The new framework we propose reflects the high degree of developmental plasticity of this neuronal population and may enable progress towards understanding of both the aetiology and treatment of metabolic disorders.

MON-709 Single-Cell RNA-Sequencing Deciphers POMC Neuron Destiny

The hypothalamus is one of the critical brain nodes regulating body weight and energy homeostasis. Within this node, Pomc neurons sense nutrient and hormonal signals to release melanocortin peptides that induce satiety, whereas AgRP/NPY neurons exert opposite effects by releasing AGRP that promotes feeding. Immature neurons in the hypothalamic ventricular zone start to express Pomc at E10.5, reach a maximum number at E14.5 and then decrease to stabilize at E18.5. However, it remains elusive how Pomc expressing precursors adopt their final cell fates. Therefore, the goal of this study was to decipher the temporal sequence of transcription factor (TF) expression leading to the terminal differentiation of POMC neurons. Red fluorescent cells collected from dissociated hypothalami of Pomc-tDimer-dsRed mice at six critical developmental time points - E11.5, E13.5, E15.5, E17.5, P5 and P12- were FACS sorted for the 10X genomics scRNA-seq pipeline. Unsupervised cell clustering identified 11 distinct clusters based on their transcriptional profiles. Eight of the clusters were highly-enriched for neuronal signature genes and were further characterized based on their transcript levels for Pomc (high, medium or low) and other distinct feature genes. Cells in the Pomchigh cluster expressed genes identified previously to modulate Pomc expression, including Isl1, Nkx2-1, and Tbx3, together with several novel candidate TFs. Unexpectedly, Nr5a1, the ventromedial hypothalamic nucleus marker gene encoding SF1, was highly expressed in the Pomchigh cluster at early stages. One of the Pomclow clusters highly expressed Otp, Agrp, Npy, Sst and Calcr while a second was highly enriched with Tac2, Kiss1, Pdyn, Prlr, Ar and Esr1 transcripts. All the clusters showed direct correlations of embryonic stage with the expression of progressively more mature markers of differentiation, thereby extending previous reports of these clusters based on single time points. Moreover, our results uncovered five novel Pomc neuron clusters with unique patterns of TF gene expression. For comparison of these data to the adult hypothalamus, we performed a TRAP-Seq study using PomcCreERT,Rosa26eGFP-L10a mice. Prdm12 and Tbx3 were among the most highly differentially expressed TFs in the POMC neuron affinity purified translatome. Similarly, Cited1, Npy2r, and Asb4 were highly expressed in both the Pomchigh cluster and the TRAP-Seq derived POMC translatome. This comprehensive molecular characterization of POMC cells during development sheds new light on the molecular diversification of early POMC neuron precursors and provides a valuable resource for elucidating the regulatory mechanisms defining POMC neuron subgroups in the hypothalamus.

NOTCH activity differentially affects alternative cell fate acquisition and maintenance.

The pituitary is an essential endocrine gland regulating multiple processes. Regeneration of endocrine cells is of therapeutic interest and recent studies are promising, but mechanisms of endocrine cell fate acquisition need to be better characterised. The NOTCH pathway is important during pituitary development. Here, we further characterise its role in the murine pituitary, revealing differential sensitivity within and between lineages. In progenitors, NOTCH activation blocks cell fate acquisition, with time-dependant modulation. In differentiating cells, response to activation is blunted in the POU1F1 lineage, with apparently normal cell fate specification, while POMC cells remain sensitive. Absence of apparent defects in Pou1f1-Cre; Rbpjfl/fl mice further suggests no direct role for NOTCH signalling in POU1F1 cell fate acquisition. In contrast, in the POMC lineage, NICD expression induces a regression towards a progenitor-like state, suggesting that the NOTCH pathway specifically blocks POMC cell differentiation. These results have implications for pituitary development, plasticity and regeneration. Activation of NOTCH signalling in different cell lineages of the embryonic murine pituitary uncovers an unexpected differential sensitivity, and this consequently reveals new aspects of endocrine lineages development and plasticity.

SOX2 is sequentially required for progenitor proliferation and lineage specification in the developing pituitary.

Sox2 mutations are associated with pituitary hormone deficiencies and the protein is required for pituitary progenitor proliferation, but its function has not been well characterized in this context. SOX2 is known to activate expression of Six6, encoding a homeodomain transcription factor, in the ventral diencephalon. Here, we find that the same relationship likely exists in the pituitary. Moreover, because Six6 deletion is associated with a similar phenotype as described here for loss of Sox2, Six6 appears to be an essential downstream target of SOX2 in the gland. We also uncover a second role for SOX2. Whereas cell differentiation is reduced in Sox2 mutants, some endocrine cells are generated, such as POMC-positive cells in the intermediate lobe. However, loss of SOX2 here results in complete downregulation of the melanotroph pioneer factor PAX7, and subsequently a switch of identity from melanotrophs to ectopic corticotrophs. Rescuing proliferation by ablating the cell cycle negative regulator p27 (also known as Cdkn1b) in Sox2 mutants does not restore melanotroph emergence. Therefore, SOX2 has two independent roles during pituitary morphogenesis; firstly, promotion of progenitor proliferation, and subsequently, acquisition of melanotroph identity.

Elimination of Kalrn expression in POMC cells reduces anxiety-like behavior and contextual fear learning

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides.Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed.

InsR/FoxO1 Signaling Curtails Hypothalamic POMC Neuron Number

Insulin receptor (InsR) signaling through transcription factor FoxO1 is important in the development of hypothalamic neuron feeding circuits, but knowledge about underlying mechanisms is limited. To investigate the role of InsR/FoxO1 signaling in the development and maintenance of these circuits, we surveyed the pool of hypothalamic neurons expressing Pomc mRNA in different mouse models of impaired hypothalamic InsR signaling. InsR ablation in the entire hypothalamus did not affect Pomc-neuron number at birth, but resulted in a 25% increase, most notably in the middle arcuate nucleus region, in young adults. Selective restoration of InsR expression in POMC neurons in these mice partly reversed the abnormality, resulting in a 10% decrease compared to age-matched controls. To establish whether FoxO1 signaling plays a role in this process, we examined POMC neuron number in mice with POMC-specific deletion of FoxO1, and detected a 23% decrease in age-matched animals, consistent with a cell-autonomous role of InsR/FoxO1 signaling in regulating POMC neuron number, distinct from its established role to activate Pomc transcription. These changes in Pomc cells occurred in the absence of marked changes in humoral factors or hypothalamic NPY neurons.

Peroxisome proliferation in POMC cells is associated with leptin resistance

Neuroendocrinology: Peroxisome proliferation in POMC cells is associated with leptin resistance

InsR/FoxO1 signaling affects neuroanatomy of hypothalamic POMC neurons

The insulin-regulated transcription factor FoxO1 functions as a major metabolic integrator in the hypothalamic feeding center and controls melanocortinergic tone. By inhibiting FoxO1, Insulin receptor (InsR) signaling regulates gene transcription in hypothalamic POMC neurons. While a role of InsR/FoxO1 signaling in the development of neuronal feeding circuits in the hypothalamus has become evident, knowledge about the underlying mechanisms remains vague. Many studies have investigated the role of the maternal environment in this context, but the role of InsR/FoxO1 signaling in hypothalamic POMC neurons themselves remains unknown. To investigate the role of InsR/FoxO1 signaling for development and maintenance of these cells, we first examined hypothalamic Pomc mRNA-expressing neurons by fluorescent in situ hybridization in mice with hypothalamic deficiency of InsR (L1). Neonatal and pre-pubertal L1 mice showed no difference in the number of Pomc-positive cells as compared to controls, suggesting that ontogeny of POMC neurons is intact in these mice. In contrast, young adult (15-week-old) L1 mice showed a 25% increase in the total number of neurons expressing Pomc, most notably in the caudal arcuate nucleus of the hypothalamus (Bregma –1.4 to –1.9mm). To determine the contribution of cell-autonomous InsR/Foxo1 function, we further generated L1 mice with selective over-activation of InsR signaling in POMC neurons (L1-Pomc+InsR), and found that the number of Pomc-positive cells in these mice was reduced by 10% compared to L1. Along this line, young adult mice with POMC-specific deletion of FoxO1 (PomcΔFoxo1) showed a 19% decrease in Pomc cell number compared to wild type controls, but again no alteration of Pomc neuron number at postnatal or pre-pubertal age. These data are consistent with a cell-autonomous role of InsR/FoxO1 in reducing POMC neuron number or silencing Pomc expression, distinct from the established role of InsR signaling in activating Pomc neuropeptide transcription through FoxO1 inhibition. Importantly, these changes in Pomc cells cannot be explained by changes in humoral factors or reciprocal changes in hypothalamic NPY neurons. Interestingly, aging is correlated with a decline in hypothalamic POMC neurons, as 30-week-old wild type mice exhibit 26% fewer Pomc-positive cells than 15-week-old mice. This aging-associated trimming of POMC neurons is not affected in L1 or PomcΔFoxo1 mice. Thus, our data support the model that InsR/Foxo1 signaling acts as a cell-autonomous gauge of ambient insulin sensitivity to direct POMC neuronal development between puberty and young adulthood.

Critical Role for Hypothalamic mTOR Activity in Energy Balance

The mammalian target of rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.

Amylin excites a subset of proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) via enhancing the excitatory glutamatergic synaptic inputs

Amylin, released from pancreas in response to nutrient ingestion, exerts anorexic effects. The ARC POMC neurons are essential for energy homeostasis by integrating a variety of peripheral and central signals that regulate energy balance. Amylin binding sites exist in the ARC. We examined the action of amylin on the ARC POMC neurons in hypothalamic slices of POMC‐EGFP transgenic mice. Bath application of amylin (1‐1000 nM) dose‐dependently increased the firing rate in 36 out of 55 POMC cells tested under loose‐patch extracellular recording. In the presence of kynurenic acid and picrotoxin to block synaptic transmission, amylin did not significantly change the firing rate of ARC POMC neurons, suggestive of an underlying presynaptic mechanism. Under voltage clamp recording, bath application of amylin (200 nM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in 7 out of 17 POMC cells, and increased the amplitude of sEPSCs in 12 out of 17 POMC cells, while increased the frequency of miniature EPSCs (mEPSCs) but decreased the amplitude of mEPSCs in 6 out of 11 POMC cells tested, respectively. These data suggest that amylin excites a subset of ARC POMC neurons via enhancing the excitatory glutamatergic synaptic inputs. (We thank Dr. Malcolm J Low for providing POMC transgenic mice and Jonathan G Murphy for help with genotyping animals, Supported by NIH grant DK62179 and CSWR foundation.)

Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

The melanocortin system as a central integrator of direct and indirect controls of food intake

the neural control of food intake and body weight is a complex function in which cognitive and emotional variables, as well as long-term signals of metabolic status and fuel storage, are integrated with short-term signals related to individual meals. Several years ago, Smith ([20][1]) proposed a

Are the silent corticotroph adenomas a new category of ACTH-producing tumours?

A minority of corticotroph adenomas are not related to Cushing's disease so that they have been defined “silent corticotropinomas“. Silent ACTH adenomas present with symptoms of mass effect, they are often haemorrhagic and show more aggressive behaviour than the conventional pituitary adenomas. Likely deriving for the POMC cells of the pars intermedia, silent corticotroph adenomas have been considered a separate entity within the spectrum of ACTH-producing adenohypophysial tumours. We studied 20 patients with non-functioning ACTH adenoma operated over the period January 1992– April 2003 at the Bellaria Hospital, Bologna, Italy. All had normal free urinary 24 hr cortisol level. Nine were female. Their age ranged from 34 to 77 years (mean, 52 years). Despite normal urinary cortisol, serum ACTH was markedly elevated in two patients (42 and 89.6 pmol/L, respectively; normal, 2–11) and slightly increased in 3. Radiologically, all lesions were macroadenoma and 12 were invasive. Cystic component was present in 5. Pathological examination revealed 8 adenomas type I, 3 of which were Crooke's cell adenomas, and 12 lesions of type II. One tumour was atypical in that it showed mitoses, MIB-1 labelling index higher that 3% and p53 nuclear over-expression. Pseudopapillary architecture due to poor cell-to-cell cohesion predominated in the majority of lesions. After a follow-up ranging from 12 to 136 months (mean, 54 months), three patients experience local recurrence but none died of disease. Silent corticotroph adenomas were compared with Cushing's related adenomas, particularly with the 13 macroadenomas operated in the same period. In these latter lesions, symptoms of mass effect were less common than in the silent tumours. Percentage of invasion was similar in the two groups whereas cystic change was more common in silent corticotropinomas. Persistence/recurrence rate was as high as noted in the silent adenoma groups. Pseudopapillary pattern was also a common finding on Cushing-related macroadenomas. Finally, we studied 5 silent corticotroph carcinomas. Patient's age ranged from 26 to 58 years. They had normal serum and free urinary cortisol as well as normal serum ACTH. All tumours were invasive macroadenoma. Histologically, 3 primary tumours were indistinguishable from conventional silent ACTH adenomas and 2 had features of atypical adenoma. All patients experienced local recurrence and, after a follow-up ranging form 2 to 23 years, all had metastases, 3 outside the central nervous system. Four patients died of disseminated disease and 1 of myocardial infarction. No patient developed Cushing's disease. Many of their clinicopathological features were similar to those of previously reported Cushing's disease related carcinomas. Our results suggested that some clinical and pathological features of silent corticotroph adenomas and carcinomas overlap with those of Cushing-related lesions. Therefore, silent adenomas are more likely part of the continuum of ACTH-producing tumours than a separate entity.

Mitochondrial uncoupling protein 2 (UCP2) in the nonhuman primate brain and pituitary.

Energy dissipating mechanisms and their regulatory components represent key elements of metabolism and may offer novel targets in the treatment of metabolic disorders, such as obesity and diabetes. Recent studies have shown that a mitochondrial uncoupling protein (UCP2), which uncouples mitochondrial oxidation from phosphorylation, is expressed in the rodent brain by neurons that are known to regulate autonomic, metabolic, and endocrine processes. To help establish the relevance of these rodent data to primate physiology, we now examined UCP2 messenger RNA and peptide expressions in the brain and pituitary gland of nonhuman primates. In situ hybridization histochemistry showed that UCP2 messenger RNA is expressed in the paraventricular, supraoptic, suprachiasmatic, and arcuate nuclei of the primate hypothalamus and also in the anterior lobe of the pituitary gland. Immunocytochemistry revealed abundant UCP2 expression in cell bodies and axonal processes in the aforementioned nuclei as well as in other hypothalamic and brain stem regions and all parts of the pituitary gland. In the hypothalamus, UCP2 was coexpressed with neuropeptide Y, CRH, oxytocin, and vasopressin. In the pituitary, vasopressin and oxytocin-producing axonal processes in the posterior lobe and POMC cells in the intermediate and anterior lobes expressed UCP2. On the other hand, none of the GH-producing cells of the anterior pituitary was found to produce UCP2. The abundance and distribution pattern of UCP2 in the primate brain and pituitary suggest that this protein is evolutionary conserved and may relate to central autonomic, endocrine and metabolic regulation.

Corticotropin-releasing factor receptor expression in the pituitary of fetal sheep after lesion of the hypothalamic paraventricular nucleus.

Both the capacity of CRF to release ACTH and the number of binding sites for CRF in the anterior pituitary decline during the final weeks of gestation in fetal sheep. The present study examined regulation of pituitary CRF receptor expression by the hypothalamic paraventricular nucleus (PVN) during late gestation in fetal sheep. Bilateral radiofrequency lesions of the PVN (PVN-Lx; n = 4) or sham lesions (SHAM; n = 5) were performed in fetal sheep at 118-122 days of gestational age (dGA). Pituitary glands from PVN-Lx and SHAM fetuses were collected at 139-142 dGA (term, approximately 148 dGA). Dual-label in situ hybridization was performed using a digoxigenin-labeled ovine POMC complementary RNA, together with a 35S-labeled ovine CRF type I (CRF1) receptor complementary RNA, to localize and quantify CRF1 receptor mRNA in POMC-hybridizing cells. Binding of [125I]-ovine CRF was also examined in the fetal pituitary of both PVN-Lx and SHAM fetuses using in situ autoradiography. The hybridization signal for the CRF1 receptor mRNA was primarily restricted to POMC-expressing cells in the anterior pituitary of both PVN-Lx and SHAM fetuses; no hybridization signal for the CRF1 receptor was observed in the neurointermediate lobe (NIL) in either group. The hybridization signal for CRF1 receptor mRNA in anterior pituitary corticotropes of PVN-Lx fetuses was significantly lower in both the inferior and superior regions of the anterior pituitary, compared with SHAM fetuses (P < 0.05). In the inferior region of the anterior pituitary, the percentage of POMC-hybridizing cells containing CRF1 receptor hybridization signal was significantly greater in PVN-Lx (90+/-7%; mean +/- SEM), compared with SHAM (67+/-6%; P < 0.05) fetuses. No differences in the percentage of POMC cells containing CRF1 receptor hybridization signal were observed in the superior region of the anterior pituitary between PVN-Lx (89+/-8%) and SHAM (87+/-9%). Binding of [125I]-ovine CRF (oCRF) was significantly greater in anterior pituitaries of PVN-Lx (140+/-19 mean arbitrary densitometry U +/- SEM), compared with SHAM (73+/-23; P < 0.05) fetuses. For both PVN-Lx and SHAM fetuses, there were no differences within group in [125I]-oCRF binding between the inferior and superior regions of the anterior pituitary. A weak, but significant (P < 0.05), autoradiographic signal for [125I]-oCRF binding was observed in the NIL of both SHAM and PVN-Lx fetal sheep. The level of [125I]-oCRF binding was significantly lower in the NIL, compared with anterior pituitary, for both SHAM (P < 0.01) and PVN-Lx fetuses. There were no differences in [125I]-oCRF binding in the NIL between SHAM and PVN-Lx fetal sheep. Our findings support a role for the PVN in regulating anterior pituitary CRF1 receptor expression in the late-gestation sheep fetus.

Effects of Photoperiod and Androgen on Proopiomelanocortin Gene Expression in the Arcuate Nucleus of Golden Hamsters**This work was supported by NIH Grants MH-44132 and K02-MH-00914 (to E.L.B.).

In golden hamsters, seasonal changes in day length act via a pineal-dependent mechanism to regulate feedback and behavioral effects of androgen. Endogenous opiates participate in photoperiodically regulated neuroendocrine functions, but the effects of androgen on expression of the gene encoding POMC, the precursor of beta-endorphin, have been controversial. We used quantitative in situ hybridization to examine regulation of POMC messenger RNA (mRNA) by testosterone and to test the hypothesis that short day lengths act through the pineal gland to amplify POMC mRNA expression. We studied intact hamsters and castrates with or without androgen treatment held in long (14 h of light, 10 h of darkness) or short (5 h of light, 19 h of darkness) days for 10 weeks. POMC gene expression differed with rostral-caudal plane, photoperiod, and surgical treatment (castration and testosterone administration). Testosterone increased the number of silver grains in labeled cells throughout the arcuate nucleus, and short day castrates given androgen consistently had more silver grains per labeled cell than did their long day counterparts. Testosterone exerted an inhibitory effect, however, on the number of POMC mRNA-positive cells, and more POMC mRNA-labeled cells were found in the arcuate nucleus of long than short day castrates treated with testosterone. Photoperiod had no significant influence in castrates not receiving androgen. Testosterone treatment had generally similar effects whether it was begun at the time of castration or 5 weeks later. Pinealectomy blocked the influence of photoperiod on both the mean number of silver grains per labeled cell and the number of labeled cells. The results indicate that day length regulates POMC gene expression when androgen levels are held constant, but that androgen is necessary for photoperiod effects to be expressed.

Effects of Photoperiod and Androgen on Proopiomelanocortin Gene Expression in the Arcuate Nucleus of Golden Hamsters

In golden hamsters, seasonal changes in day length act via a pineal-dependent mechanism to regulate feedback and behavioral effects of androgen. Endogenous opiates participate in photoperiodically regulated neuroendocrine functions, but the effects of androgen on expression of the gene encoding POMC, the precursor ofβ -endorphin, have been controversial. We used quantitative in situ hybridization to examine regulation of POMC messenger RNA (mRNA) by testosterone and to test the hypothesis that short day lengths act through the pineal gland to amplify POMC mRNA expression. We studied intact hamsters and castrates with or without androgen treatment held in long (14 h of light, 10 h of darkness) or short (5 h of light, 19 h of darkness) days for 10 weeks. POMC gene expression differed with rostral-caudal plane, photoperiod, and surgical treatment (castration and testosterone administration). Testosterone increased the number of silver grains in labeled cells throughout the arcuate nucleus, and short day castrates given androgen consistently had more silver grains per labeled cell than did their long day counterparts. Testosterone exerted an inhibitory effect, however, on the number of POMC mRNA-positive cells, and more POMC mRNA-labeled cells were found in the arcuate nucleus of long than short day castrates treated with testosterone. Photoperiod had no significant influence in castrates not receiving androgen. Testosterone treatment had generally similar effects whether it was begun at the time of castration or 5 weeks later. Pinealectomy blocked the influence of photoperiod on both the mean number of silver grains per labeled cell and the number of labeled cells. The results indicate that day length regulates POMC gene expression when androgen levels are held constant, but that androgen is necessary for photoperiod effects to be expressed.

Pituitary hyperplasia.

Comprehensive article summarizing more than 25 years of experience with pituitary hyperplasia in surgical material. Morphologic forms of hyperplasia--diffuse and nodular--are defined and, for comparison, the normal morphology, frequency and intraglandular distribution of cell types are briefly reviewed. All cell types can give rise to hyperplasia, although their frequency, extent and clinical importance widely vary. Somatotroph hyperplasia is rare; it is limited to cases of GHRH overproduction by extrapituitary endocrine neoplasms and sporadic examples of gigantism. Prolactin cells display the highest propensity for non-neoplastic proliferation. Physiologic hyperplasia occurs in pregnancy and lactation. Pathological hyperplasia is mostly secondary to other, neoplastic or non-neoplastic, space occupying processes. Idiopathic lactotroph hyperplasia is very rare. The much-disputed corticotroph hyperplasia is infrequent cause of pituitary dependent Cushing's disease. Despite difficulties of diagnosis in fragmented biopsies, several well-documented cases prove the existence of corticotroph hyperplasia which is nearly always nodular. Thyrotroph hyperplasia, secondary to hypothyroidism, a treatable condition, is not expected to occur in surgical material, yet several cases have been identified. Operated lesions are massive nodular leading to significant pituitary enlargement thereby mimicking TSH- or PRL-producing adenoma. Hyperprolactinemia is a frequent concomitant of severe thyrotroph hyperplasia. Gonadotroph hyperplasia and proliferation of pars intermedia derived POMC cells are not likely to occur in surgical material and have no clinical significance. Adenoma formation may rarely be associated with any type of pituitary hyperplasia.