Morphological, histological and ultra-structural studies on the ultimobranchial body of goat (Capra hircus).

Ultimobranchial body (UBB) remnant was considered as an enigma till the last few years, then it was recognized as a necessary organ where it is the origin of the parafollicular cells. The samples were fixed and processed for the histological and electron microscopic examination. Macroscopically, the UBB remnant appeared as a white mass at the end of the cranial one-third of the thyroid lobe. It was composed of solid cell nest, cluster of cells and small thyroid follicles. Transmission electron microscope showed some round cells containing euchromatic nuclei, numerous parafollicular cells with darkly stained granules and paler ones. It also showed some mast cells with heterochromatic nuclei and large darkly stained granules. The parafollicular cells were distributed throughout the thyroid gland but concentrated within the UBB remnant. To our knowledge, our findings represent very unique histological manifestations specially the ultra-structural ones which revealed an original finding about the new clear type of cells suggested to be a UBB remnant and ensure those of the light microscope.

Positivity for GATA3 and TTF-1 (SPT24), and Negativity for Monoclonal PAX8 Expand the Biomarker Profile of the Solid Cell Nests of the Thyroid Gland.

Solid cell nests (SCNs) are usually distinguished on conventional H&E-stained sections; however, the morphological heterogeneity in SCNs and hyperplasia of these ultimobranchial body remnants can mimic other diagnostic entities including but not limited to papillary microcarcinoma. In order to confirm the thyroid follicular epithelial origin and exclude the possibility of SCNs, most diagnosticians use immunohistochemical biomarkers of thyroid follicular epithelial cells and/or those of SCNs. While the expression profile of monoclonal PAX8 has not been reported previously in SCNs, the status of TTF-1 expression using the 8G7G3/1 clone has been inconsistent among several studies. Given the potential diagnostic pitfalls, this series investigated the expression profile of GATA3, monoclonal PAX8, and TTF-1 (SPT24), along with p63, p40, monoclonal calcitonin, monoclonal CEA, and HBME-1 in a tissue microarray (TMA) of 56 SCNs. SCNs were all diffusely and strongly positive for TTF-1 (SPT24), p63, and p40, and were negative for monoclonal PAX8 and calcitonin. Positivity for GATA3 and monoclonal CEA was identified in 41 (73.2%) and 36 (64.3%) of SCNs. In addition, 18 (32.1%) SCNs displayed HBME-1 reactivity. These findings expand the immunohistochemical correlates of SCNs by demonstrating positivity for GATA3 and TTF-1 (SPT24), and negativity for monoclonal PAX8. The identification of monoclonal CEA expression and HBME-1 in SCNs also underscores the limitations of these select biomarkers in the distinction of C cell proliferations and papillary microcarcinoma, respectively. The findings of this series also suggest that positivity for TTF-1 (SPT24) alone should not be used to confirm the thyroid follicular epithelial origin. Therefore, the combined use of TTF-1 (SPT24) and monoclonal PAX8 in association with p63 or p40 provides an accurate distinction of SCNs.

Immunohistochemical profiling of the ultimobranchial remnants in the rat postnatal thyroid gland.

Ultimobranchial (UB) remnants are a constant presence in the thyroid throughout rat postnatal life; however, the difficulty in identifying the most immature forms from the surrounding thyroid tissue prompted us to search for a specific marker. With that objective, we applied a panel of antibodies reported to be specific for their human counterpart, solid cell nests (SCNs), using double immunohistochemistry and immunofluorescence. Our results demonstrated that cytokeratin 34ßE12 and p63 are highly sensitive markers for the immunohistologic screening of UB-remnants, independently of their maturity or size. Furthermore, rat UB-follicles (UBFs) coincided with human SCNs in the immunohistochemical pattern exhibited by both antigens. In contrast, the pattern displayed for calcitonin and thyroglobulin differs considerably but confirm the hypothesis that rat UB-cells can differentiate into both types of thyroid endocrine cells. This hypothesis agrees with recent findings that thyroid C-cells share an endodermic origin with follicular cells in rodents. We suggest that the persistence of p63-positive undifferentiated cells in UB-remnants may constitute a reservoir of basal/stem cells that persist beyond embryogenesis from which, in certain unknown conditions, differentiated thyroid cells or even unusual tumors may arise.

Nerium indicum, a botanical pesticide affects ultimobranchial gland of the catfish Heteropneustes fossilis.

Heteropneustes fossilis were subjected to 11.27 mg L(-1) (80% of 96 h LC50 ) and 2.81 mg L(-1) (20% of 96 h LC50 ) of Nerium indicum leaf extract for short-term and long-term, respectively. After sacrificing the fish, blood was collected on 24, 48, 72, and 96 h in short-term and after 7, 14, 21, and 28 days in long-term experiment and analyzed for plasma calcium levels. Also, ultimobranchial glands (UBG) were fixed on these intervals. Serum calcium levels of H. fossilis exhibited a decline after 48 h following exposure to Nerium indicum leaf extract. This decrease continued till the end of the experiment (96 h). Ultimobranchial cells exhibited a decrease in the cytoplasmic staining response after 72 h following the treatment. The nuclear volumes of these cells were slightly decreased. These changes were exaggerated after 96 h following the treatment. Chronically exposed fish exhibited a decline in serum calcium levels of H. fossilis on day 14. The level progressively declined till the end of the experiment. Up to day 14 following the treatment there was no change in the histological structure of UBG. A decrease in the nuclear volume of ultimobranchial cells was noticed on day 21. Moreover, the cytoplasm of these cells displayed weakstaining response. The nuclear volume of these cells recorded a further decrease following 28-day treatment. Also there was noticed vacuolization and degeneration at certain places. To the best of our knowledge, the effects of any botanical pesticides on fish UBG have not been reported yet.

Development of thyroid gland and ultimobranchial body cyst is independent of p63.

The ultimobranchial body (UBB) and thyroid primordium are the origins of the thyroid gland that fuse around embryonic day 14.5 of mouse gestation, ultimately giving rise to calcitonin-producing C cells and thyroglobulin-producing follicular cells, respectively. A homeodomain transcription factor NKX2-1 is expressed both in the UBB and the thyroid primordium, and is critical for development of the thyroid gland. In this study, the role of p63 in development of UBB and the thyroid gland was analyzed by histological, immunohistochemical, and electron microscopic analyses using mice with various combinations of Nkx2-1 and p63 wild-type, heterozygous, and null alleles. In the absence of p63, a normal thyroid gland develops, as revealed by expression of thyroglobulin and calcitonin, thus showing that p63 is not required for thyroid development. However, in mice carrying the Nkx2-1-null allele, the UBB remains as a cystic vesicular structure and/or in nested patterns consisting of p63-positive cells surrounding the vesicle and undifferentiated immature cells with occasional cilia lying inside. The cystic UBB was present even in the Nkx2-1;p63 double-null mice. The structure and p63 expression pattern of the UBB cyst strikingly resemble the solid cell nest. These results show that in the absence of NKX2-1, UBB becomes cystic independent of p63, which is likely the origin of SCN.

Histological alterations in the ultimobranchial gland of teleost Heteropneustes fossilis in response to chlorpyrifos treatment.

In this study, an experiment was performed on Heteropneustes fossilis for short-term (1.76 mg/L chlorpyrifos, i.e., 0.8 of 96-h LC50) and long-term (0.44 mg/L chlorpyrifos, i.e., 0.2 of 96-h LC50) exposure. The fish were sacrificed after 24, 48, 72 and 96 h in the short-term experiment and after 7, 14, 21 and 28 days in the long-term experiment. On these intervals, blood was collected and analysis of serum calcium was done. Ultimobranchial glands were also fixed for histological study. The serum calcium levels of H. fossilis exhibit a decline after 24 h following exposure to chlorpyrifos. This decrease continues until the end of the experiment (96 h). The serum calcium levels of chronically exposed fish exhibit a decrease on day 7. Thereafter, the levels continue to fall progressively until the end of the experiment (28 days). The ultimobranchial gland of chlorpyrifos treated fish exhibits no histological change up to 48 h. After 72 h, there is a decrease in the staining response of cytoplasm of the ultimobranchial cells. The nuclear volume of these cells is slightly decreased. After 96 h following chlorpyrifos exposure, these changes become exaggerated. In chlorpyrifos-treated fish there is no change in the histological structure of the ultimobranchial gland up to 14 days. After 21 days, the cytoplasm of ultimobranchial cells stain feebly and the nuclear volume of these cells exhibits a decrease. Following 28 days treatment, the nuclear volume of these cells records a further decrease and the gland depicts vacuolization and degeneration at certain areas.

Mash1 regulates the development of C cells in mouse thyroid glands.

In mammals, the ultimobranchial body derived from the fourth pharyngeal pouch gives rise to thyroid C cells. The C cells of newborn mice are immunoreactive for calcitonin, calcitonin gene-related peptide (CGRP), protein gene product (PGP) 9.5 and NeuroD, and transiently exhibit the neuronal markers TuJ1 and somatostatin during fetal development. The basic helix-loop-helix (bHLH) transcription factor Mash1 plays a role in the differentiation of autonomic neurons. We show that in wild-type mouse embryos, Mash1 is expressed in the ultimobranchial body at embryonic day (E) 12.5, when the body is located close to the great arch arteries. It is also expressed in the ultimobanchial body fused with the thyroid lobe at E 13.5. Targeted disruption of Mash1 resulted in the absence of C cells in the mouse thyroid glands, since cells displaying the C-cell markers and expressing NeuroD were not detected during fetal development or at birth. The failure of C-cell formation in the null mutant thyroids was also confirmed by electron microscopy. While the formation and migration of the ultimobranchial body were not affected in the Mash1 null mutants, at E 12.5-E 13.5 both the ultimobranchial body located close to the arteries and the organ populating the thyroid lobe exhibited a marked increase in apoptotic cell numbers. Thus, in the mutant mice, the ultimobranchial body fails to complete its differentiation program and finally dies. These results indicate that Mash1 enhances survival of the C-cell progenitors by inhibiting apoptosis.

Calcitonin is expressed in the chicken pituitary gland: influence of gonadal steroids and sexual maturation.

Calcitonin (CT) is primarily produced by the thyroid C cells in mammals or by the ultimobranchial gland in chickens. CT is also expressed by the pituitary gland in rats in which it functions as a paracrine factor causing decreased lactotroph proliferation and prolactin (PRL) secretion. Gonadal steroids influence CT expression in the rat pituitary gland. However, the expression of the CT gene in the pituitary gland of chickens or of any other avian species has not previously been reported. We have tested the hypotheses that CT is expressed in the chicken pituitary gland, and that its expression is influenced by sexual maturation or in response to ovarian steroid administration. We have detected robust expression of CT cDNA in the chicken pituitary gland by reverse transcription/polymerase chain reaction (PCR). The sequence of the pituitary-derived CT cDNA is identical to that of the ultimobranchial gland. CT-immunoreactive (ir) cells have been observed throughout the anterior pituitary gland by confocal microscopy. Many of the PRL-ir cells show co-localization with CT-ir cells. Quantitative real-time PCR analysis has revealed an inverse relationship between the quantities of PRL mRNA and CT mRNA in the pituitary gland: sexually mature hens contain lower amounts of CT mRNA but larger quantities of PRL mRNA compared with sexually immature chickens. Estradiol and/or progesterone treatment of sexually immature chickens leads to a significant decrease in the quantity of pituitary CT mRNA relative to that in the vehicle-treated chickens. We conclude that pituitary CT plays an important paracrine/autocrine role in the control of lactotroph function and PRL secretion in the chicken.

Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1 expression is required for development and fusion of the ultimobranchial body to the thyroid.

The ultimobranchial body (UBB) is an outpocketing of the fourth pharyngeal pouch that fuses with the thyroid diverticulum, giving rise to calcitonin-producing C-cells. In this study, we demonstrate that the UBB is composed of two types of cells: one expressing T/ebp/Nkx2.1 and the other expressing p63. The former cell type, accounting for a majority of the UBB, requires T/ebp/Nkx2.1 for their survival. In contrast, the p63-positive cells, even in the absence of T/ebp/Nkx2.1 expression, can proliferate and give rise to a vesicular structure that is lined by a monolayer of p63-negative cells, surrounded by a cluster and/or single layer of p63-positive cells, displaying the basal/stem cell phenotype. T/ebp/Nkx2.1 haploinsufficiency causes abnormal fusion of the UBB with the thyroid diverticulum, which stays as a cluster of C-cells around the vesicular structure, similar to the one observed in mice null for T/ebp/Nkx2.1 expression. These results demonstrate that T/ebp/Nkx2.1 plays a role in the survival of UBB cells, their dissemination into the thyroid diverticulum, and the formation of UBB-derived vesicular structure.

Possible direct induction by estrogen of calcitonin secretion from ultimobranchial cells in the goldfish.

The plasma level of calcitonin (CT), a calcium (Ca)-regulating hormone, is known to increase in female teleosts during the reproductive period. In the present study, a correlation between plasma CT and Ca and one between plasma CT and the gonad somatic index were demonstrated in the female goldfish but not in the male. To clarify the relationship between CT and Ca, we examined the plasma CT and Ca levels after injecting immature goldfish with estrogen. At day 1, the plasma CT level significantly increased, whereas the plasma Ca level was not changed from its initial level. This result suggests that the trigger of CT secretion is estrogen and that estrogen directly acts on the ultimobranchial gland (UBG), a CT-secreting organ. To determine whether the UBG is equipped with estrogen receptor (ER), an ER binding assay and immunohistochemical staining of UBG cells with an antibody against ER were conducted. As a result, estrogen-specific binding (Kd, 18.52 nM; Bmax, 1.35 pmol/mg protein) and ER-immunoreactivity in the UBG were demonstrated. Furthermore, the expression of alpha, beta, and gamma types of ER in the UBG was also detected by use of the reverse-transcription polymerase chain reaction. Thus, we concluded that estrogen acts on the UBG to induce the release of CT, which in turn plays an important role in reproduction directly and/or indirectly through Ca. This is the first report on the existence of ERs in a teleost UBG and the occurrence of CT secretion caused by estrogen.

Analysis of salmon calcitonin I in the ultimobranchial gland and gill filaments during development of rainbow trout, Oncorhynchus mykiss, by in situ hybridization and immunohistochemical staining.

We have cloned two distinct cDNAs encoding salmon-type calcitonin (sCT)-I cDNAs from the ultimobranchial gland of rainbow trout, Oncorhynchus mykiss. Both cDNAs were predicted to encode nearly identical sCT-I precursors which consisted of an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-I, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. Development of sCT-I-expressing cells was then examined by employing conventional histochemical staining, in situ hybridization with a specific cRNA probe, and further immunohistochemistry. The primordium of the ultimobranchial gland was first identified, as two cell masses, in the region between the alimentary canal and sinus venosus behind the heart 17 days postfertilization (dpf; 14 degrees C). However, expression of sCT-I mRNA could not be detected in this gland until one day later, and appeared at 18 dpf. sCT-I immunoreactivity was first observed at 19 dpf (two days before hatching), and the ultimobranchial gland began to assume a follicular structure at 20 dpf (one days before hatching). As ontogeny proceeded, the sCT-I-immunoreactive cells increased in both number and stainability. The sCT-I mRNA was also expressed on the developing gill filaments, but immunoreactive sCT-I was not detected in these sites. These results provide basic data for further research on the organogenesis of the trout ultimobranchial gland.

Expression and localization of prohormone convertase PC1 in the calcitonin-producing cells of the bullfrog ultimobranchial gland.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the ultimobranchial gland of the adult bullfrog using immunohistochemical (IHC) and in situ hybridization (ISH) techniques. In the ultimobranchial gland, PC1-immunoreactive cells were columnar, and were present in the follicular epithelium. When serial sections were immunostained with anti-calcitonin, anti-CGRP, anti-PC1, and anti-PC2 sera, PC1 was found only in the calcitonin/CGRP-producing cells. No PC2-immunopositive cells were detected. In the ISH, PC1 mRNA-positive cells were detected in the follicle cells in the ultimobranchial gland. No PC2 mRNA-positive cells were detected. RT-PCR revealed expression of the mRNAs of PC1 and the PC2 in the ultimobranchial gland. However, very little of the PC2 mRNA is probably translated because no PC2 protein was detected either by IHC staining or by Western blotting analysis. We conclude that the main prohormone convertase that is involved in the proteolytic cleavage of procalcitonin in the bullfrog is PC1.

Dynamics of plasma Ca and calcitonin levels in stonefish (Inimicus japonicus) administered a high-Ca solution into the stomach.

In stonefish, changes in plasma total Ca and calcitonin levels were examined after administration of a high-Ca solution into the stomach. Blood was taken successively at 0, 1, 3, 9, 33, and 81 hr from a fine tube cannulated into the aortic bulb. Plasma Ca levels increased acutely at 1 hr and attained the peak after 3-9 hr of the administration. Although plasma calcitonin levels did not exhibit conspicuous changes for 1-3 hr, they began to rise significantly at 33 hr. The plasma Ca level began to decline significantly at 33 hr, although the level was still significantly higher than the initial level. At 33 hr, however, the plasma calcitonin level still continued to increase. At 81 hr, the plasma Ca level had returned to the initial level. At that time, the plasma calcitonin level was also significantly lower than that at 33 hr. These results suggest that, in stonefish, the ultimobranchial gland has the ability to respond physiologically to rises in plasma Ca levels, to secrete calcitonin, and to cease the secretion when the plasma Ca levels return to the initial level.

Direct raises in blood Ca levels by infusing a high-Ca solution into the blood stream accelerate the secretion of calcitonin from the ultimobranchial gland in eels.

In eels, a CaCl(2) solution was infused into the pneumatic duct vein. Plasma Ca levels were significantly increased during 3 hr and were followed by significant raises in plasma calcitonin levels. These results strongly suggest that, in eels, direct raises in blood Ca levels by infusion of a high-Ca solution via blood vessels can accelerate the secretion of calcitonin from the ultimobranchial gland.

Neuronal properties in cultured ultimobranchial C cells of chick embryos: process outgrowth and expression of TuJ1 and enkephalin.

We have analyzed the neuronal properties in cultured ultimobranchial C cells isolated from embryonic chicks at different developmental stages (12--16 days of incubation) by immunohistochemistry and electron microscopy. The ultimobranchial glands mostly consist of C cell solids. In 13-day-old embryos, many C cells cultured for 7 days on laminin-coated slides extended long neurite-like processes, reaching 300 microm in length. Neuritic outgrowth of C cells was regulated developmentally and virtually unaffected by nerve growth factor (NGF). The cultured C cells expressed intense immunoreactivity for calcitonin and enkephalin. It was also confirmed by confocal laser-scanning microscopy that almost all C cells were intensely immunostained by both the calcitonin antiserum and the monoclonal antibody TuJ1, a neuron-specific marker. Scanning electron microscopy identified the outgrowth of long, branching neuritic processes emerging from C cell soma. The processes had numerous varicosities along their course and ended in growth cones. The C cells with processes were usually monopolar and less frequently bipolar or multipolar. Transmission electron microscopy revealed the presence of membrane-bounded secretory granules in the cultured C cells. The neuritic processes of C cells contained aggregations of microfilaments, intermediate filaments and microtubules arranged in parallel to the long axis. In addition, synaptic-like contacts showing desmosome-like membrane-thickenings and accumulations of small clear vesicles and dense-cored vesicles were formed between the endings of the processes and the surface of C cells. These results indicate that the C cells cultured from early chick embryos (12- and 13-day-old) maintain the neuronal characteristics for long periods in vitro.

Ultrastructural features of "solid cell nest" of the human thyroid gland: a study of 8 cases.

The ultrastructural features of solid cell nests (SCN), made of squamous cells, and associated calcitonin cells (C cells), of the thyroid gland were studied in only a few cases in humans. A study was performed on 8 paraffin-embedded SCN, postembedded in Epon, to look for their ultrastructural features. Immunohistochemical analysis using calcitonin antibody was performed on semithin sections of SCN to explore the presence of C cells. Three cases (37.5%) of SCN were positive for calcitonin, and electron-dense secretory granules were observed in the cytoplasm. In two of these cases, an increased number of C cells in the adjacent thyroid parenchyma was observed. The presence of ciliated and lymphoid cells, in addition to intracytoplasmic microvacuolar and microfollicular (microglandular) structures, was noticed. Ciliated cells have already been reported in embryonic rests of human and animals, but ultrastructurally for the first time in human SCN. The presence of microfollicular structures, intracytoplasmic microvacuolar, secretory granules features, and ciliated cells, in addition to lymphoid cell, suggests the existence of a common ultimobranchial stem cell for C cells or for one or more cell types of the thyroid gland.

[Calcitonin and stanniocalcin. Particular aspects of the endocrine regulation of phospho-calcium metabolism in mammals and fish]. / Calcitonine et stanniocalcine. Particularités de la régulation endocrinienne du métabolisme phospho-calcique chez les mammifères et les poissons.

The hypocalcemic and hypophosphatemic peptide calcitonin (CT) is secreted by mammalain thyroid parafollicular cells and fish ultimobranchial body. Over a dozen species of CTs have been cloned and/or sequenced. They can be separated into three classes based on structural and biological similarities: teleost/avian, artiodactyl, and human/rat. In mammals, CT exerts its anti-hypercalcemic and hypophosphatemic effects by inhibiting osteoclastic bone resorption and renal tubular phosphate reabsorption, respectively. CT receptors (CTRs) are members of a subfamily of seven-transmembrane domain, G protein-coupled receptors that include those for several other peptide hormones. Basic amino acid substitutions within the CT molecule enhance potency, probably by conferring a helical structure to the peptide. This might explain the enhanced potency of fish CTs for mammalian CTRs. The presence and secretion of salmon CT-like immunoreactive material have been described in both the murine and human central nervous systems, which possess CTRs. These findings are consistent with a role for this peptide acting as a neurotransmitter in mammals. Stanniocalcin (STC) is another hypocalcemic hormone originally identified in fish. In fish STC exerts its anti-hypercalcemic effect by regulating calcium and phosphate transports by the gills, intestine and kidney. Although fish ultimobranchial cells are much less responsive to the secretagogic effects of Ca2+ than mammalian parafollicular cells, the secretion of both CT and STC are positively regulated by extracellular calcium. STC has also been recently identified in humans and rats. It is released by some renal tubular cells and might play a role in the regulation of phosphate metabolism. Nevertheless, the true physiologic roles for CT in fish and STC in mammals, respectively, remain unknown.

"Aging bull'.

An old bull, it is said by those who know, can have his troubles. Included among these are vertebral osteosclerosis and ankylosing spondylosis; this stiffening up limits, rather than accentuates, the value and reproductive potential of a stud bull past his prime. Associated with these abnormalities, however-and not seen in age-matched cows of comparable breeds-are fascinating endocrine neoplasms suggestive of a pattern that could be productive as a model of human hereditary endocrine abnormalities. Adjacent to the thyroid gland in other vertebrates are ultimobranchial bodies that are incorporated into the lateral thyroid lobes in primates as the parafollicular "C cells' of the thyroid. These are the cells in man that give rise to medullary thyroid cancer and are associated with calcitonin secretion, useful as a tumor marker. In aging bulls of whatever breed, nearly half exhibit abnormality of these ultimobranchial bodies: 20% show hyperplasia, and 30% have frank neoplasia. These ultimobranchial tumors appear in bulls passing 6 1/2 years in age, and are absent in young bulls and all cows of any age. Calcitonin can be demonstrated in the ultimobranchial tumors from bulls, and secretion is stimulated by calcium infusion, though serum calcium remains normal. The ultimobranchial tumors themselves can range from hyperplasia through adenoma to metastasizing carcinoma-in fact, representing one of the commoner cattle cancers. Parathyroid glands taken from bulls with these ultimobranchial tumors initially show evidence of inhibited secretory activity and morphologic atrophy, but later go on to develop hyperplasia and, eventually, autonomy. Cattle forage on calcium-rich diets. Bulls appear to respond to this calcium excess from the positive balance, but breeding cows have the unique calcium deficits of the high net loss of calcium through lactation and the large requirements of calcifying a fetal skeleton. Chronic stimulation of the APUD-derived ultimobranchial bodies by high calcium intake, not counterbalanced by calcium losses in the bulls, may account for the development over time of the ultimobranchial neoplasms. Further, a number of the bulls who have the ultimobranchial tumors are found to have multiple endocrine tumors in other glands-bilateral pheochromocytomas and pituitary acidophil adenomas.

Estrogen receptors in the stingray (Dasyatis akajei) ultimobranchial gland.

On the basis of our previous observation that estrogen stimulates calcitonin secretion by the ultimobranchial gland in the stingray, Dasyatis akajei, experiments were conducted to examine the presence of estrogen receptor (ER) and its mRNA in the gland, employing exchange assay and Northern blot analysis, respectively. The optimal incubation conditions of the exchange assay for cytosolic ER were found to be 25 degrees for 2 hr. Scatchard analysis of cytosolic ER revealed two components with dissociation constants (Kd) of 0.090 +/- 0.015 and 17.780 +/- 1.910 nM and with a maximum number of binding sites (Bmax) of 62.2 +/- 7.9 and 2290 +/- 180 fmol/mg protein, respectively. Furthermore, Sephadex G-100 filtration of the cytosol and immunoblot analysis using antiserum against a recombinant rat ER confirmed the presence of two specific binding components for estrogen (41 and 57 kDa). Total RNA extracted from the ultimobranchial gland was subjected to Northern blot analysis using a rat ER cDNA as a probe. Two positive signals were detected at 2.5 and 2.0 kb. Thus, the presence of ER was confirmed for the first time in nonmammalian calcitonin-secreting cells.

Production of salmon calcitonin I in Oncorhynchus gorbuscha by alternative polyadenylation of two RNA species.

RNAs of ultimobranchial bodies (U.B.) from the pink salmon, Oncorhynchus gorbuscha, were studied using the polymerase chain reaction (PCR) with specific oligodeoxyribonucleotides (oligos) of the salmon calcitonin (sCT) mRNA selected in exon 2 or 3 and a poly(T) oligo. We observed two amplified DNA fragments, differing by 200 bp which hybridized with a specific exon 4 probe. Sequence analysis indicated that they both encoded exon 4, but differed in the length of their 3' non-coding regions by use of a putative polyadenylation signal situated 200 bp upstream from the established polyadenylation site. These two polyadenylation signals very likely were regulated differently, as the larger expressed transcript was predominant. To date, such use of an alternative polyadenylation signal in a CT mRNA has not been described in other vertebrates, and only the chicken CT mRNA possesses a second classical polyadenylation signal which is not known to be used. This characteristic of sCT biosynthesis appears to be typical in lower vertebrates and is of phylogenic interest. Moreover, it engenders a hypothesis of a relationship between the high concentration of the peptide observed in females of this species and their capacity to produce sCT by different biosynthetic pathways.