The sheep pars tuberalis: an immunohistochemical study. Demonstration of the presence of glycoprotein and lipotropin hormones

Summary Using indirect immunofluorescence with fourteen different antisera raised against pituitary hormones and peptides, we characterized immunochemically the cells of the sheep pars tuberalis. The presence of LH-and FSH-containing cells, shown in previous studies, was also observed in the present investigation. In addition, we found TSH-containing cells, never observed in sheep, and LPH-containing cells. The latter hormone has never been found in any studied species. It appeared that a small amount of perikarya (less than 20%) were immunolabelled and, that the sheep pars tuberalis contained a majority of immunonegative cells as in the guinea-pig, rabbit and rhesus monkey. This study may contribute to a better knowledge of the function of the sheep pars tuberalis.List of abbreviations ACTH adrenocorticotropin hormone - BSA bovine serum albumin - CGRP calcitonin gene-related peptide - FSH follicle stimulating hormone - GH growth hormone - HSA human serum albumin - LH luteinizing hormone - LH-RH luteinizing hormone-releasing hormone - LPH lipotropin hormone - Met-enk methionine enkephalin - NPY neuropeptide Y - POMC proopiomelanocortin - PRL prolactin - TSH thyreotrope stimulating hormone     

Cellular composition of the human pituitary pars tuberalis as revealed by immunocytochemistry

Summary An attempt was made to determine if any of the specialized secretory cell types common to the pars distalis also occur in the pars tuberalis of the human hypophysis. Available for study were 18 specimens of the inferior pars tuberalis, which partially surrounds the infundibular stem, and 3 specimens of the superior pars tuberalis that is attached to the median eminence. Antisera to human somatotropin, mammotropin, chorionic gonadotropin, follicle-stimulating hormone, FSH , luteinizing hormone, LH , thyrotropin, TSH , as well as to ^1–24-corticotropin, porcine ^17–39-corticotropin, and ovine LH were used with the Sternberger peroxidase-antiperoxidase immunocytochemical procedure to identify the probable cells of origin for these hormones.The evidence indicated that gonadotropic cells constitute the major portion of the parenchymal cell population in the pars tuberalis. They occurred throughout all of the pars tuberalis and were usually arranged in clusters. Somatotropic, mammotropic, corticotropic, and thyrotropic cells were rare and not found in all specimens. When present, they often formed a common group suggesting that their occurrence in the pars tuberalis resulted from displacement of primordial tissue of the pars distalis during embryogenesis.Supported in part by research grants HD-03159 and HD-08333 from the National Institute for Child Health and Human DevelopmentWe thank Dr. L.A. Sternberger for providing the PAP complex and others for antisera (Table 2) and hormones (Footnote 2) as listed     

Regulation of DNA methyltransferase in the testis of rat

In mammalian DNA, the base 5-methylcytosine is generated by post-replicational methylation of cytosine residue by DNA methyltransferase. In this study the levels of DNA methyltransferase of testis during various ages and the effects of gonadotropic hormones on immature rat testis were studied. It was observed that the specific activity of DNA methyltransferase was high in the testis at the age of 20 and 30 days. After this age, the activity of DNA methyltransferase declined significantly and was maintained at lower level from days 120 to 240. Treatment with follicle stimulating hormone significantly decreased the enzyme activity in the testis while luteinizing hormone did not cause any effect. These results indicate that DNA methyltransferase of certain cells in the testis is under the influence of follicle stimulating hormone.     

Determination of releasing hormones in subcellular fractions from porcine hypothalamic tissue

In a study of possible biosynthesis of hypothalamic hormones in mitochondria, the activities of subcellular fractions from gradient Ficoll fractionation were assayed. Mitochondrial fractions identified by qualitative assays for oxygen uptake and phosphorylation, showed both respiratory activity and release of the luteinizing hormone. Purification of solvent extracts of mitochondrial fractions by Bio-Gel P-2 and Sephadex G-25 yielded fractions which released the luteinizing and follicle stimulating hormones and somatotropin. Labeled pGlu-His-Pro-NH₂ and the release of thyrotropin served as a control.     

Differential regulation of DNA methylation in rat testis and its regulation by gonadotropic hormones

Eukaryotic DNA methylation occurs exclusively at the 5'-position of cytosine and has been implicated in the regulation of gene expression. Using high-performance liquid chromatography, the methylation of testis DNA during its development, in different cell populations and during regulation by gonadotropic hormones, were studied. The 5-mC content of testis DNA increased significantly from days 30 to days 150, while in 2-yr-old testis 5-mC content decreased significantly. Among various populations of testicular cells, pachytene spermatocyte DNA contained a significantly high amount of 5-mC when compared to spermatogonia, spermatids and mature sperm DNA. However, the 5-mC content of elongated spermatids was significantly less when compared to the above four fractions. Administration of follicle stimulating hormone to immature rats caused hypomethylation of seminiferous tubular DNA while luteinizing hormone caused similar effects in Leydig cells. These results indicate that in testis, DNA methylation is differentially regulated during development and is controlled by gonadotropic hormones.     

Fine Structure of the Pars Distalis of the Pituitary Gland in the Female Mink,Mustela vison

Pituitary glands of intact and experimental adult females of mink, Mustela vison, were examined by electron microscopy. Conventional methods involving removal of endocrine glands (ovaries and adrenals), administration of radioactive isotope, ¹³¹I, blocking agents (thiouracil and metopirone) and hormones (thyroxine, hydrocortisone, thyrotropin and luteinizing hormone releasing hormones) were employed. Five categories of granular cells were distinguished both by their ultrastructural characteristics and qualitative changes throughout the year and following different treatments. The cell types are described and their functions discussed. From conventional electron microscopical studies it proved difficult to draw any satisfactory conclusions about the gonadotropic cells. Further investigation by means of immunocytochemistry and radioimmunoassay techniques is required to determine, whether the presumptive gonadotropic cell type produces both FSH and ICSH or only one of these hormones. Morphologically two types of agranular cells were identified. Their morphological inter-relationship and function are discussed briefly.     

Intermediate filament expression in non-neoplastic pituitary cells.

Fifty-one non-neoplastic human pituitary glands, including examples with Crooke's hyalinization or amyloidosis, were examined by an immunoperoxidase method using antibodies to keratin, vimentin, neurofilaments (NFs), glial fibrillary acidic protein (GFAP), desmin, actin, S-100 protein and a variety of pituitary hormones. It was confirmed that most of the epithelial cells in the pituitary gland express keratin immunoreactivity. These cells included endocrine cells in the anterior lobe, endocrine cells and squamous metaplastic cells in the pars tuberalis, columnar and ciliated epithelia forming follicular structures and salivary-type epithelium in the pars intermedia, and anterior lobe cells infiltrating the posterior lobe. This study also demonstrated that keratin and NFs may be co-expressed in endocrine cells in the pituitary anterior lobe, that keratin, vimentin and GFAP may be co-expressed in the epithelial cells forming cyst-like follicle in the pars intermedia, and that vimentin and GFAP may be co-expressed in folliculo-stellate cells and pituicytes. In addition, the GFAP and S-100 protein-negative high columnar epithelium in the pars intermedia tended to be positive for adrenocorticotropic hormone and melanocyte stimulating hormone, while the low columnar epithelium with the co-expression of GFAP and S-100 protein was negative for pituitary hormones.     

Hypophysiotropic activity of histone H3 in vitro

To assess the effect of histone H3 on pituitary hormone secretion, rat anterior pituitary (AP) cells were used and growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle stimulating hormone measured by radioimmunoassay. Incubation of cells with H3 (1, 6, and 30 microM) stimulated the release of all five hormones in a dose-dependent manner. This effect was blocked by preincubation of H3 with an anti-H3 antibody. Incubation of AP cells with 6 microM H3 in the presence of specific AP hormone secretagogues (GRP-6, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH)) showed additive effects on hormone secretion. Pharmacological experiments suggested that calcium- and diacylglycerol- (DAG) associated pathways, but not cAMP, participate in the hypophysiotropic activity of H3. Our results confirm previous evidence that histones may act as hypophysiotropic signals.     

Pituitary gonadotropins regulate spermatogonial differentiation and proliferation in the rat‡

The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle stimulating hormone in the spermatogonial proliferation has been studied using specific antibodies against these hormones in the immature rats. Immunoneutralization of lu teinizing hormone for 7 days resulted in significant reduction in tetraploid cells and total absence of haploid cells,while there was a relative increase in the diploid population. This was also accomopanied by a decrease in spermatogonial proliferation as indicated by a decrease in [³H] thymidine incorpor-ation into DNA by purified spermatogonia. Administration of follicle stimulating hormone a/s for 7 days also caused a significant decrease in the rate of spermatogonial proliferation. Withdrawal of follicle stimulating hormone led to a significant reduction in tetraploid and haploid cells. However interestingly,it failed to totally abolish the appearance of these cells. Administration of testosterone (3 mg/day/rat) for 2 days along with the gonadotropin a/s could partially reverse the effect on spermatogonial proliferation. It is concluded that (i) both luteinizing hormone and follicle stimulating hormone are involved in spermatogonial proliferation, (ii) lack of testosterone consequent of the neutralization of luteinizing hormone prevented the entry of spermatogonial cells into meiosis, (iii) testosterone may be involved in spermatogonia] proliferation providing a mitotic signal and (v) both follicle stimulating hormone and testosterone act synergistically and lack of any one of the hormones results in impairment of spermatogonial proliferation. A part of the data was presented at the 16th International Congress of Biochemistry and Molecular Biology, New Delhi, September 1994.     

Intercellular communications within the rat anterior pituitary. XVI: Postnatal changes of distribution of S-100 protein positive cells,connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5–60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis.     

Demonstration of gonadotropic cells in the adenohypophysis (pars distalis and pars tuberalis) of the monkey Macacus irus. Immunofluorescence study using human anti-beta-FSH and ovine anti-beta-LH antibodies

Indirect immunofluorescence technique with anti-beta FSH and anti-beta oLH antisera has allowed us to detect "gonadotropic cells" in the pars distalis and in the pars tuberalis of the adenohypophysis of the monkey Macacus irus. In the pars distalis, 85-90 % of the "gonadotropic cells" react simultaneously with these two antisera ; 10-15 % of these cells react only either with anti-beta hFSH or anti-beta oLH antisera. The gonadotropic cells are dispersed in the whole pars distalis, amid the other cellular types ; indeed, in the female, there is a "gonadotropic zone" in the median zone of the lateral lobes of the gland. In the pars tuberalis, we have observed "gonadotropic cells" which react only with anti-beta oLH antiserum. These results are compared with observations of some authors.     

Localization and development of chromogranin A and luteinizing hormone immunoreactivities in the secretory-specific cells of the hypophyseal pars tuberalis of the chicken

 The pars tuberalis mainly consists of the secretory cells specific to this portion of the pituitary. We examined the localization and development of luteinizing hormone (LH) and chromogranin A in the chicken pars tuberalis by immunohistochemistry. The vast majority of the chicken pars tuberalis was occupied by cells immunoreactive for both LH and chromogranin A. Furthermore, immunoblot analysis of chicken pars tuberalis extracts with LH antiserum demonstrated that two bands, the large α-subunit and small β-subunit of the LH molecule, were expressed in this tissue as well as in the pars distalis. A band for chromogranin A was also detected in pars tuberalis extracts with chromogranin A antiserum. In contrast to the cells of mammalian species that contain only a few small secretory granules, the specific cells of the chicken pars tuberalis were characterized by the presence of many secretory granules ranging from 90 to 400 nm in diameter. Postembedding immunogold labeling showed that gold particles representing immunoreactivity for LH were densely located on all secretory granules of the secretory-specific cells. Many secretory granules, especially the large ones, of the cells were also loaded with immunogold particles for chromogranin A. Double immunogold labeling confirmed that LH and chromogranin A were colocalized on the same secretory granules. During embryonic development, the primordium of the pars tuberalis was first detected at 8 days of incubation as a small group of cells containing LH- and chromogranin-immunoreactive cells. In the pars distalis, the onset of LH and chromogranin expression occurred earlier, at 6 days of incubation. At 10 days of incubation, the pars tuberalis primordium became large cell masses consisting of LH- and chromogranin-immunoreactive cells, which were located close to the median eminence. Subsequently, the primordium extended along the median eminence progressively with age. At 14 days of incubation, it reached to the rostral end and surrounded the median eminence as slender cell cords. These results indicate that specific cells of the chicken pars tuberalis synthesize a glycoprotein hormone related to the LH molecule, which is stored in the secretory granules together with chromogranin A. The pars tuberalis may be involved in the regulation of gonadal function in a different way from that of the pars distalis. Accepted: 26 August 1997     

Histology of the pituitary gland of the grey mullet, Mugil cephalus L.

The present communication deals with a histological study of the pituitary gland of the teleost fish Mugil cephalus , found in the estuarine waters of Cochin area. Six different cell types were identified in the pituitary gland on the basis of their grouping, distribution and staining properties. The prolactin and the TSH cells (thyroid stimulating hormone producing cells or thyrotrops) were identified in the rostral pars distalis and the ACTH cells (adrenocorticotropic hormone producing cells or corticotrops) in the interphase between the neurohypophysis and the rostral pars distalis. The STH cells (somatotropic hormone producing cells or somatotrops) and the gonadotropic cells were distinguished in the proximal pars distalis and the MSH cells (melanin stimulating hormone producing cells or melanotrops) in the pars intermedia.     

Unchanged response to stimulation of pituitary hormone release after serial UV irradiation in men

A group of 24 healthy young men were evaluated before and after serial suberythematous ultraviolet (UV) radiation: group I, control (no irradiation); groups II and III, 12 radiations in 4 weeks with two different spectra (both containing UV-B). Before the first and 2 days after the last exposure all the volunteers were given an intravenous injection of thyrotropin releasing hormone (TRH, protirelin 0.2 mg) and luteinizing hormone releasing hormone (LH-RH, gonadorelin 0.1 mg). The serum concentrations of TSH, follicle stimulating hormone, LH and prolactin were measured at 0, 20, 30, 45 and 60 min by radioimmunoassay. Neither basal nor stimulated levels of the pituitary hormones showed significant changes after UV radiation. The results showed that exposure to suberythematous doses of UV did not influence the regulation of pituitary hormones in these healthy individuals. Accepted: 24 October 1996     

The sheep pars tuberalis: an immunohistochemical study. Demonstration of the presence of glycoprotein and lipotropin hormones

Using indirect immunofluorescence with fourteen different antisera raised against pituitary hormones and peptides, we characterized immunochemically the cells of the sheep pars tuberalis. The presence of LH- and FSH-containing cells, shown in previous studies, was also observed in the present investigation. In addition, we found TSH-containing cells, never observed in sheep, and beta LPH-containing cells. The latter hormone has never been found in any studied species. It appeared that a small amount of perikarya (less than 20%) were immunolabelled and, that the sheep pars tuberalis contained a majority of immunonegative cells as in the guinea-pig rabbit and rhesus monkey. This study may contribute to a better knowledge of the function of the sheep pars tuberalis.     

Desensitization of testicular ornithine decarboxylase to gonadotropin releasing hormone and gonadotropic hormones

Prior exposure of the testis to gonadotropin releasing hormone, luteinizing hormone or follicle stimulating hormone caused the testis refractory to these hormones in terms of ornithine decarboxylase activity at 24 h. Luteinizing hormone caused desensitization in the Leydig cells while the levels of ornithine decarboxylase in the seminiferous tubules were unaltered. In gonadotropin releasing hormone desensitized testis all the other treated compounds namely, luteinizing hormone, follicle stimulating hormone, prostaglandin F2 alpha, norepinephrine and cyclic AMP caused stimulation of ornithine decarboxylase activity. The testis desensitized with LH responded to cyclic AMP and norepinephrine whereas prostaglandin E2 or gonadotropin releasing hormone caused less stimulation of ornithine decarboxylase activity. These results indicate that testicular desensitization to gonadotropin releasing hormone and luteinizing hormone is not due to a post cyclic AMP block.     

Occurrence of colloid-containing follicles and ciliated cysts in the hypophysial pars tuberalis from guinea pigs of various ages

Colloid-containing follicles and ciliated cysts in the hypophysial pars tuberalis of guinea pigs at various ages ranging from 5 days to 36 months were examined by periodic acid-Schiff (PAS) reaction, immunohistochemistry, and electron microscopy. The follicles storing PAS-positive colloid were encountered in the pars tuberalis of all guinea pigs examined, although only a few were present in young animals. The follicles gradually increased in number with age. The largest number of follicles was found in the senile male group: 141.3 +/- 11.9, about 10 times the number in the 5-day-old male group. The follicles were scattered throughout the entire length of the pars tuberalis. Follicles with enlarged luminal cavities were concentrated in the ventral caudal region surrounding the infundibular stem and merges with the pars distalis. Three different types of follicles were found by electron microscopy: 1) those surrounded by nongranulated follicular cells that may correspond to the stellate-follicular cells in the pars distalis, 2) those surrounded by specific cells that were packed with vesicular inclusions, and 3) those surrounded by granulated cells that may be gonadotropes. In the follicles lined by non-granulated follicular cells, long, prominent microvilli and cytoplasmic processes protruding into the lumen and invaginations of colloid were often observed at the apical cell region. The follicles lined by the specific cells having numerous vesicles were localized only in the ventral caudal portion. The vesicles ranged from 200 to 700 nm in diameter, and the outer surface of their limiting membrane was partly studded with ribosomes. Gonadotropes immunoreactive to the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) antisera were distributed in the guinea pig pars tuberalis. As well as the typical follicles described above, the follicles composed solely of granulated cells showed microvilli protruding into the cavities and junctional complexes at the apical lateral surface. They stored heterogeneous materials in the lumina. Some secretory granules gave the appearance of being discharged into the lumen. Ciliated cysts were frequently observed in the pars tuberalis; their incidence was 71.7%. The ciliated cysts were much larger than colloid-containing follicles. Cystic cavities were only partly filled with heterogeneous materials showing colloid-like, flocculent, and granular features.     

Effect of diethylstilbesterol and prolactin on the induction of follicle stimulating hormone receptors in immature and cycling rats

Induction of follicle stimulating hormone receptor in the granulosa cells of intact immature rat ovary by diethylstilbesterol, an estrogen, has been studied. A single injection of 4 mg of diethylstilbesterol produced 72 h later a 3-fold increase in follicle stimulating hormone receptor concentration as monitored by [¹²⁵I]-_oFSH binding to isolated cells. The newly induced receptors were kinetically indistinguishable from the preexisting ones, as determined by Lineweaver-Burk plot of the binding data. The induced receptors were functional as evidenced by increased ability of the granulosa cells to incorporate [³H]-leucine into cellular proteins. Neutralization of endogenous follicle stimulating hormone and luteinizing hormone by administering specific antisera had no effect on the ability of diethylstilbesterol to induce follicle stimulating hormone receptors, whereas blockade of endogenous prolactin secretion by ergobromocryptin administration significantly inhibited (∼ 30 %) the response to diethylstilbesterol; this inhibition could be completely relieved by ovine prolactin treatment. However, ovine prolactin at the dose tried did not by itself enhance follicle stimulating hormone receptor level. Administration of ergobromocryptin to adult cycling rats at noon of proestrus brought about as measured on diestrusII, (a) a reduction of both follicle stimulating hormone (∼ 30 %) and luteinizing hormone (∼ 45 %) receptor concentration in granulosa cells, (b) a drastic reduction in the ovarian tissue estradiol with no change in tissue progesterone and (c) reduction in the ability of isolated granulosa cells to convert testosterone to estradiol in response to follicle stimulating hormone. Ergobromocryptin treatment affected only prolactin and not follicle stimulating hormone or luteinizing hormone surges on the proestrus evening. Treatment of rats with ergobromocryptin at proestrus noon followed by an injection of ovine prolactin (1 mg) at 1700 h of the same day completely reversed the ergobromocryptin induced reduction in ovarian tissue estradiol as well as the aromatase activity of the granulosa cells on diestrus II, thus suggesting a role for proestrus prolactin surge in the follicular maturation process     

Ontention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicle stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropins and development of its radioimmunoassay

Using the classical approach, a decapeptide was synthesized with the structure of porcine luteinizing hormone/follicle stimulating hormone releasing hormone reported by Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V. (1971) Biochem. Biophys. Res. Commun. 43, 1393–1399. As already reported, this peptide was capable of inducing in vitro the release of luteinizing hormone and follicle stimulating hormone from rat pituitary glands. A specific antiserum against luteinizing hormone/follicle stimulating hormone releasing hormone has been generated in the guinea pig and this allowed the development of a radioimmunoassay for this peptide. The antisera, at a final dilution of to depending on the antiserum used, were able to bind 35% of the ¹³¹I-labelled antigen. The sensitivity of this assay method was 50 pg of luteinizing hormone/follicle stimulating hormone releasing hormone. The following substances did not cross-react: oxytocin, lysine-vasopressin, synthetic thyroid stimulating hormone releasing hormone, ovine luteinizing hormone, follicle stimulating hormone and prolactin. Des-Trp³ luteinizing hormone/follicle stimulating hormone releasing hormone, pyroglutamyl-histidyl-tryptophan and seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycinamide, exhibited flatter curves than luteinizing hormone/follicle stimulating hormone releasing hormone with a cross-reactivity of about . Using this method, luteinizing hormone/follicle stimulating hormone releasing hormone was assayed in extracts of the sheep stalk-median eminence and of the hypothalamus and in jugular vein blood from a normal ram and from normal male rats, from cyclic ewe and from hypophysectomized ram and rats. It was concluded that luteinizing hormone/follicle stimulating hormone releasing hormone is present in hypothalamic extracts and in plasma of sheep and rat.     

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog,Rana limnocharis

Cells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross-react with α-MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross-reactivity.