Ultrastructure of rat pituitary LH gonadotrophs in relation to serum and pituitary LH levels following repeated LH-RH stimulation

The effects of single and repeated LH-RH injections at 120 min intervals on female rat LH gonadotrophs and on pituitary and serum LH levels were investigated using electronmicroscopy and radioimmunoassay. A temporary stimulation of granule release, of protein and new granule synthesis and of the accumulation of lysosomal structures was found in LH cells after the first LH-RH injection. The temporary stimulations were massively enhanced after the second injection. These consecutive yet in their time-sequence overlapping processes account for the initial depletion of secretory granule content (3--15 min after LH-RH injection), for the subsequent regranulation and accumulation of granules above control levels (60--120 min after injection) and also for the reduction in the number of granules to control levels (150 min after LH-RH injection and thereafter). Increased polymorphic lysosomal structures are believed to be responsible for this reduction of excess granules. The amount of assayable pituitary and serum LH generally corresponds with the morphological changes observed in LH-gonadotrophs, thus further substantiating the above observations. A schema which summarizes the observed morphological and hormonal changes in their time-sequence in response to LH-RH stimulation depicts the short-term regulation of secretory processes in female gonadotrophs.     

Thyroidectomy cells and their response to thyrotrophin releasing hormone (TRH) in the rat

Summary Thyroidectomy cells of the rat pituitary gland were studied by the peroxidase-antibody labeling procedure and by electron microscopy. Secretory granules accumulated in these cells in response to a short-term treatment with thyroxine, and the cells were then reactive to the peroxidase-antibody labeling procedure. An intravenous injection of synthetic thyrotrophin releasing hormone (TRH) to thyroxine-treated, thyroidectomized rats provoked an acute and active extrusion of secretory granules from the thyroidectomy cells. The secretory granules in these cells were mostly haloed after primary fixation in osmium tetroxide. It is concluded that TRH causes thyroidectomy cells to release their secretory granules, and presumably TSH, by the usual process of exocytosis or granule extrusion.This study was supported by USPHS Grant AM 12583.     

Stereological and biochemical analysis of prolactin and growth hormone secreting rat pituitary cells in culture

Summary The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH₁2C₁, GH₃ and GH₄C₁), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with ³H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed, the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. Alle three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH₄C₁), implying that the increase of prolactin secretion, at lest in part, is due to increased prolactin synthesis.     

Effects of gonadotropin-releasing hormone (GnRH) on the cytodifferentiation of gonadotropes in rat adenohypophysial primordia in organ culture

The effects of gonadotropin-releasing hormone (GnRH) on the development of gonadotropes were investigated by the use of organ culture and by means of immunocytochemistry and radioimmunoassay. Pituitary primordia from rat fetuses were cultured in a medium with or without 10^-9 M GnRH during the first 24 h of culture. The ratio of the number of immunoreactive LH cells to the total number of cells in the explants derived from 13.5-day fetuses was increased by the GnRH treatment after 6 or 8 days of culture, while the total number of cells was not altered. LH released into the medium and LH content of explants were not affected by the GnRH treatment. Subsequent treatment with 10^-9 M GnRH for 4 h after 7 days of culture resulted in a marked release of LH, accompanying a significant decline in LH content, in both explants exposed or unexposed to the first GnRH treatment. However, the former explants contained a lower amount of LH than the latter explants. The present results indicate that pituitary primordia at 13.5 days of gestation are capable to respond to GnRH, and that GnRH is effective in stimulating the responsiveness of gonadotropes to GnRH during early pituitary cytodifferentiation.     

Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture

Summary Semi-thin sections of three-dimensional reaggregates from adult female rat pituitary, cultured in serum-free defined medium, were stained for prolactin, gonadotropin, thyrotropin, growth hormone and S-100, using the double immunolabelling technique. The frequency of juxtaposition between lactotrophs and gonadotrophs was enumerated and compared with the expected frequency at random distribution of polygonal cell profiles in a hexagonal configuration. The proportions of lactotrophs and gonadotrophs in the aggregate sections were determined using stereometrical analysis. The observed frequency of juxtaposition did not differ significantly from the expected frequency. Hence, no reason was found to assume a selective adhesion between lactotrophs and gonadotrophs in adult female rat pituitary reaggregates. A constant proportion of lactotrophs was found to meet the criteria of a cup-shaped morphology, and 70%±9% (mean ±S.D.) of these so-called cupshaped lactotrophs were found to be juxtaposed at their concave side to gonadotrophs. Administration of 0.01 nM 17-oestradiol to the culture medium resulted in a significant reduction of the proportion of cup-shaped lactotrophs but did not affect the selectivity of juxtaposition to gonadotrophs. The selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs may be the morphological correlate of the functional relationship between these cells, which are known to be involved in an intra-pituitary paracrine communication system.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Distribution of 7B2 (secretogranin V)-like immunoreactivity in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary

In this study, we examined 7B2 (secretogranin V)-like immunoreactivity (IR) in the Japanese red-bellied newt (Cynops pyrrhogaster) pituitary. Results showed that the pars nervosa was filled with immunoreactive granules. In the pars intermedia, all melanotrophs showed 7B2-IR. In the pars distalis, immunoreactive cells were dispersed, and the 7B2-immunoreactive cells were also immunopositive for the β-subunit of bullfrog luteinizing hormone (fLHβ). 7B2-IR co-localized with fLHβ-IR in the same secretory granules. Our results suggest that 7B2 may participate in the secretion processes of gonadotropins in the pars distalis.     

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat

Summary To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2–60 min) after intravenous injection of mono-radioiodinated ¹²⁵I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp⁶, Pro⁹ (Net), DesGly¹⁰]-GnRH produced comparable results. In contrast, the injection of ¹²⁵I-[D-pGlu¹, D-Phe², Trp^3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.     

LHRH inactivation by reconstituted horse and fetal bovine sera: assessment by reduction of immunoreactivity and biological activity in pituitary cell cultures

Lyophilized horse and fetal bovine sera are commonly incorporated into the growth media used for primary pituitary cell cultures. LHRH degrading activity has been assumed to exist in these preparations but has not actually been demonstrated. During our studies with pituitary cultures, it became necessary to ascertain if LHRH inactivating activity could be demonstrated in these sera. Luteinizing hormone releasing hormone (LHRH) was preincubated in either serum-free medium or medium containing fetal bovine and horse serum. Whether LHRH was lost during these incubations was assessed by diminished immunoreactivity as indicated by radioimmunoassay (RIA) and by diminished biological activity as indicated by reduced release of LH from pituitary cell cultures. Both the RIA and bioassay results indicated LHRH inactivating activity; the loss of LHRH could be prevented by inclusion of bacitracin in the incubations.     

The origin and ultrastructural characteristics of corticotropin-releasing factor (CRF)-immunoreactive nerve fibers in the posterior pituitary of the rat

Summary A fine network of corticotropin-releasing factor (CRF)-immunopositive fibers was found in the posterior lobe of the pituitary of the rat. The intermediate and distal lobes were free of CRF-immunoreactivity. Varicose, terminal-like axons were frequently observed around capillary vessels. Surgical isolation of the paraventricular nuclei resulted in a complete disappearance of CRF-immunoreactive fibers from the posterior lobe. CRF-immunopositive fibers show the general characteristics of peptidergic axons. These ultrastructural observations support the idea that CRF is secreted into capillary vessels.     

Effects of Neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat

Neuropeptide Y (NPY) has been recently localized in several hypothalamic nuclei in the mammalian brain. In order to investigate the possible role of NPY on neuroendocrine function, we have investigated the effects of the peptide on the release of anterior pituitary hormones in the rat. Both intravenous (300 μg) or intraventricular (2 to 15 μg) injection of NPY produced in gonadectomized male rats a significant and long-lasting decrease of plasma LH levels. A short duration stimulating effect on prolactin plasma levels was also observed after the intravenous but not after the intraventricular injection of NPY. Plasma levels of the other pituitary hormones were not significantly modified after NPY injection. When incubated in vitro with anterior pituitary cells in monolayer culture, NPY produced no significant change in release of pituitary hormones. Thus NPY seems to exert a selective effect on LH release. Since this effect can be observed after both intravenous and intraventricular injection, it might be hypothesized that NPY could affect LHRH release in two areas which lack blood-brain barrier: the organum vasculosum of the lamina terminalis (OVLT) which contains LHRH cell bodies and NPY fibers and the median eminence which contains both LHRH and NPY fibers. The effect on prolactin release needs to be carefully evaluated in different experimental conditions.     

Gonadotropin-releasing hormone stimulates annexin 5 messenger ribonucleic acid expression in the anterior pituitary cells

We previously reported that annexin 5 is found specifically in gonadotropes and that the expression is dramatically enhanced after ovariectomy. In the present study, the expression of annexin 5 was examined in the primary culture of rat anterior pituitary cells using semiquantitative RT-PCR to determine if it is under the direct control of gonadotropin-releasing hormone (GnRH). Continuous administration of GnRH analog for 1 h enhanced the expression of both FSH beta subunit and annexin 5 mRNA. The expression of annexin 5 mRNA was also augmented by phorbol 12-myristate 13-acetate but not by forskolin. Administration of recombinant rat annexin 5 to the culture increased LH beta mRNA expression. These data clearly demonstrate that the expression of annexin 5 mRNA is directly controlled by GnRH and suggest that annexin 5 is involved in mediating GnRH action in the pituitary gland.     

Orexin-B-like immunoreactivity localized in both luteinizing hormone- and thyroid-stimulating hormone-containing cells in the Nile tilapia (Oreochromis niloticus) pituitary

Immunohistochemical techniques were employed to examine orexin-like immunoreactivities in the pituitary of the Nile tilapia (Oreochromis niloticus). Rabbit anti-orexin-A serum and mouse anti-orexin-B monoclonal antibodies were used as primary antibodies. Orexin-B immunoreactive cells corresponded to luteinizing hormone (LH)- or thyroid-stimulating hormone (TSH)-containing cells, and all LH- and TSH-containing cells were immunoreactive for orexin-B. However, we found no orexin-A immunoreactive cells in the pituitary. In the Nile tilapia, an orexin-B-like substance may be secreted from LH- or TSH-containing cells and may regulate pituitary function, rather than the orexin-A-like substance in the pituitaries of Japanese seaperch and medaka.     

Effects of colchicine on release of neurosecretory material from the posterior pituitary gland of the rat

Summary The effect of colchicine on the release of neurosecretory material from the posterior pituitary gland was investigated in the rat in vivo and in vitro. Colchicine was administered subarachnoidally when neurophysin, radiolabelled by injection of (³⁵S) cysteine into the supraoptic nucleus, had accumulated in the neural lobe. Dehydration for 3 days of non-colchicine-treated rats was followed by a 100% reduction of neurophysin-bound radioactivity. When colchicine was given prior to dehydration, the reduction of radioactive neurophysin was less marked. Colchicine treatment alone was likewise followed by a lowering of protein-bound radioactivity in the neural lobe, which may indicate that colchicine, in addition to blocking the rapid axonal transport of neurosecretory material, also impedes the slow transport.The release of radioactive neurophysin in response to depolarizing concentration of potassium in vitro was diminished in the presence of colchicine, the reduction being most pronounced after colchicine treatment in vivo. The biochemical data prove the view that colchicine inhibits the release of neurosecretory material from the neural lobe. The ultrastructural findings support the biochemical data. Thus, colchicine treatment alone or followed by dehydration induced a marked increase in the number of organelles, especially of mitochondria and dense bodies. There was a marked increase in the number of enlarged axons filled with dammed organelles in the infundibulum and neurohypophysis. There was an accumulation of dense core vesicles and microvesicles in the axonal terminals in the neurohypophysis after treatment with either colchicine or colchicine followed by dehydration, which indicates an impediment of the release process. Dehydration alone induced a depletion of the dense core vesicles in the terminals. Out from the combined biochemical and ultrastructural findings possible mechanisms for the action of colchicine are discussed.The present study was supported by grants from Svenska livförsäkringsbolags fond för medicinsk forskning, The Swedish Medical Research Council (No. B73-12X-2543-05B), Magnus Bergvalls stiftelse and from the Medical Faculty, University of Göteborg.Miss Gull Grönstedt is thanked for careful secretarial work and the technical assistance by Mrs. Wally Holmberg, Mrs. Elisabeth Norström and Mrs. Ulla Svedin is gratefully acknowledged. Abbreviations used: NSG = neurosecretory granules; NSN = neurosecretory material; SON = supraoptic nucleus.     

Effect of intracerebroventricular infusion of leptin on the secretory activity of the GnRH/LH axis in fasted prepubertal lambs

Leptin is believed to link metabolic status to reproductive processes. The aim of the present study was to investigate the effect of exogenous leptin on the secretory activity of GnRH/LH system in acutely undernourished prepubertal, female lambs. Merino lambs were randomly divided into four groups, two standard-fed and two fasted for 72 h. One standard and one fasted groups were infused intracerebroventricularly (i.c.v.) with the vehicle; the remaining standard and fasted groups were infused with leptin (25 μg/120 μl/h). Leptin was administered in series of four 1-h infusions at 30-min intervals for 3 consecutive days from 08:30 to 14:00 h. Blood samples were collected on day 0 (before infusions) and on day 3 every 10 min over a 6-h period. Immediately after the experiment, the sheep were slaughtered and brains fixed in situ. Hypothalamic and pituitary tissues were prepared for further immunohistochemical and hybridization in situ analysis. In fasted sheep, increased GnRH levels in the median eminence (P < 0.001) and LHβ levels in the pituitary cells (P < 0.001) plus decreased LHβ mRNA and LH pulsatility in blood plasma were observed (P < 0.05). In leptin-infused fasted sheep, GnRH levels in the median eminence decreased (P < 0.001), LHβ mRNA hybridization signal increased, LHβ levels decreased in the pituitary cells (P < 0.001) and LH pulsatility increased (P < 0.05) in the blood plasma. These results indicate that, in prepubertal sheep, the GnRH/LH axis is sensitive to the fasting signal, that influence of which can be reversed by leptin. Leptin cancels out the suppressing effect of fasting on LH secretion by augmentation of GnRH.     

Localization of microfilaments in prolactin cells of the rat anterior pituitary gland

Summary Prolactin cells from anterior pituitary glands of normal non-lactating female rats, and lactating animals, some of which were separated from their pups for 48 hours, were examined ultrastructurally for the presence of microfilaments. Microfilaments were found in specific intracellular locations in all cells examined. They were in association with the nuclear envelope, the Golgi complex, the endoplasmic reticulum, small vesicles of the endoplasmic reticulum, and secretory granules. The possible role of microfilaments in the movement of intracellular organelles is considered.This investigation was supported by the National Institutes of Health grants AM 12583 and TW 02023.The authors wish to express their gratitude to Mr. M. G. Williams and Miss Pauline Cisneros for their excellent technical assistance.     

LH responses to single doses of exogenous GnRH in the Cape mole rat (Georychus capensis): the pituitary potential for opportunistic breeding

The Cape mole rat Georychus capensis is a solitary mole rat that inhabits the winter rainfall region of the Western Cape Province of South Africa. Circulating basal concentrations of luteinizing hormone (LH) were found to be significantly higher in the breeding season in both sexes. During both the breeding and non-breeding season, administration of exogenous gonadotropin-releasing hormone (GnRH) increased circulatory LH levels. The magnitude of the LH response to an overdose of exogenous GnRH both in and out of the breeding season in males and females was not significantly different. Typically, seasonally breeding species exhibit a down-regulation of the pituitary and reproductive functions out of the breeding season. It appears that there is no down-regulation of GnRH receptors at the level of the pituitary out of the breeding season, because the pituitary responds to an exogenous GnRH challenge equally both in and out of the breeding period. The Cape mole rat exhibits the potential for opportunistic breeding out of the breeding period, provided that environmental factors are favourable. This finding questions whether this mole rat is actually a seasonal breeder or whether reproduction is hindered by the ecological constraint of the lack of opportunities to burrow and find mates at certain times of the year.     

Granulated ‘marginal cell layer’ in the rat anterior pituitary gland

A granulated ‘marginal layer cell’ was observed in the lining of Rathke's residual pouch of 5 and 10 day-old rat anterior pituitary glands. Immunohistochemistry was not employed to identify the precise function of these cells. However, the cytological characteristics of nearly all of the cells indicated that they resembled GH-secreting cells, with a few displaying morphological features of corticotrophs. In pituitary glands of 5–20 day-old rats, both ends of Rathke's residual pouch extended into the pars distalis at the site of transitional zone of this lobe and of the pars intermedia. The cells within the ‘invading’ residual pouch contained numerous microvilli. In the middle portion of the residual pouch, cavities lined by ‘marginal layer cells’ had numerous microvilli and were adjoined by junctional complexes. In the adult rat pituitary gland, there were no granulated cells in the ‘marginal cell layer’ and no invasion of the residual pouch into the anterior lobe. From these data the possible source of the follicle and of the folliculo-stellate cells in the anterior pituitary of the rat is proposed.