ARTICLE |
Correspondence to: M. Carmen González, Dept. of Anatomy, Faculty of Medicine, University of La Lagna, La Laguna, Tenerife, Spain. E-mail: tgonhern@ull.es
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The presence of neuronal nitric oxide synthase (nNOS) in two populations of pituitary cells, gonadotrophs (LH) and folliculostellate (FS) cells, suggests that pituitary nitric oxide (NO) is involved in the control of hormone secretion. We have used single and double immunostaining and quantitative procedures to investigate possible gender-related differences in the nNOS expression pattern in the anterior pituitary lobe and its possible alterations in different endocrine situations. Our results reveal a sexual dimorphism in the pattern of nNOS expression. In males, nNOS is mainly found in FS cells, whereas only a few LH cells express nNOS. Conversely, in females, nNOS is mainly found in LH cells. After gonadectomy, paralleling an increase in LH cell size and serum luteinizing hormone (LH) levels, there is nNOS upregulation in LH cells and nNOS downregulation in FS cells. After testoterone replacement, LH cells become nNOS-immunonegative again. In lactating rats, LH cells overexpress nNOS, but LH cell size and serum LH levels are low. This suggests that, depending on its cellular source, pituitary NO can exert either an inhibitory or a stimulatory effect on hormone secretion. When released from FS cells, NO exerts a paracrine inhibitory effect, and when released from gonadotrophs it exerts an autocrine or paracrine stimulatory effect on LH or prolactin secretion, respectively. (J Histochem Cyotochem 48:16391647, 2000)
Key Words: nitric oxide, pituitary gland, gonadotrophs, folliculostellate cells, gonadectomy, lactation, rat
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NITRIC OXIDE (NO) is a short-lived free radical involved in a wide range of biological functions, including regulation of vascular tone, cell-mediated immune responses, and cellular signaling in the brain (
With immunohistochemical and in situ hybridization techniques, it has been possible to map the distribution of NOS in various tissues, including the endocrine system. In the posterior lobe of the pituitary gland, nNOS has been demonstrated in axon terminals that come from magnocellular hypothalamic neurosecretory neurons ( in FS cells in anterior pituitary cultures (
Functional studies suggest that NO may be involved in the secretion of different pituitary hormones (
With the aim of contributing to a better understanding of the functional meaning of NO in the pituitary gland, and particularly in LH secretion, we have investigated possible gender differences in the pattern of nNOS activity under normal conditions, and the effect of different endocrine manipulations on its expression, by using immunocytochemical and double immunofluorescence techniques. In addition, we used quantitative analysis of the number and size of LH cells (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and Tissue Preparation
Male and female SpragueDawley rats (IFFA-Credo; Lyon, France) weighing 260280 g were studied. The following endocrine models were used: male rats castrated 15 days beforehand (n = 5), castrated with testosterone replacement for 3 days (1.7 mg/d; n = 4), and intact male rats (n = 5); and ovariectomized (n = 5), lactating female (15 days after delivery; n = 4), and female rats in the diestrous stage of the estrous cycle (n = 5). Surgical interventions were performed under aseptic conditions, and all animals used in the research described here were acquired and cared for in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and the principles presented in the "Guidelines for the Use of Animals in Neuroscience Research" by the Society for Neuroscience.
Rats were heavily anesthetized with chloral hydrate and transcardially perfused with 150 ml of heparinized 0.9% saline and 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). Before perfusion, blood samples of 1 ml were obtained from anesthetized animals for subsequent determination of serum LH levels, using a rat luteinizing hormone EIA system kit supplied by Amersham (Poole, UK). Pituitary glands were removed and stored in the same fixative at 4C for 2 hr. After cryoprotection in 30% sucrose in PB overnight at 4C, 20-µm-thick coronal sections were cut with a freezing microtome. Sections were collected in three parallel series, one for single NOS immunostaining, another for either LH (as a marker of gonadotrophs), prolactin (as a marker of lactotrophs) or S-100 (as a marker of FS cells) single immunocytochemistry, using 3,3-diaminobenzidine (DAB) as chromogen, and the third one for either NOS/LH or LH/prolactin double immunofluorescence, or single NOS, prolactin, or S-100 immunofluorescence. Bearing in mind that fixative conditions are critical in the histochemical and immunocytochemical detection of NOS- expressing cells (
Immunocytochemistry
For single immunolabeling, sections were immersed for 20 min in 3% H2O2 to inactivate endogenous peroxidase and incubated for 60 min at room temperature (RT) in the preincubation solution (PIS): 4% normal horse serum (NHS; Vector Laboratories, Burlingame, CA) or 4% normal goat serum (NGS; Vector Laboratories) in 0.1 M PBS, pH 7.4, containing 0.1% Triton X-100 (TX-100; Sigma, St. Louis, MO). Thereafter, they were incubated overnight in a gently shaking humid chamber in PIS containing one of the following primary antibodies. For nNOS, we used a rabbit anti-nNOS polyclonal antibody (1:2000; Zymed Laboratories, San Francisco, CA); for LH, a mouse anti-human ß3LH monoclonal antibody (1:3000; Serotec, Oxford, UK); for prolactin, a rabbit anti-rat prolactin polyclonal antibody (1:3000; a gift from Dr. A.F. Parlow, UCLA Medical Center, Torrance, CA); and for S-100, a rabbit anti-S-100 polyclonal antibody (1:800; Sigma). After several rinses, sections were incubated for 2 hr in biotinylated horse anti-mouse antiserum (1:200; Vector) or biotinylated goat anti-rabbit antiserum (1:200; Vector) and 1:200 NHS or 1:200 NGS in PBS. Immunoreactions were visible after incubation for 1 hr at RT in ExtrAvidinperoxidase (1:5000; Sigma) in PBS, and after 10 min in 0.005% DAB and 0.001% H2O2 in cacodylate buffer. After several rinses in PBS, the slides were dehydrated, cleared in xylene, and coverslipped with DPX (BDH Chemicals; Poole, UK).
For immunofluorescence, sections were immersed for 60 min in PIS and overnight in PIS containing one of the primary antibodies used in immunocytochemistry at double concentration. For double immunofluorescent staining, we followed the same procedure, but the incubation contained a mixture of two of the primary antibodies. The immunofluorescent labeling was visible after incubation for 3 hr in 1:100 fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Sigma) and 1:150 lissaminerhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch; West Grove, PA) in PBS containing 1:200 NGS (Vector). After several rinses, sections were air-dried, coverslipped with Vectashield (Vector), and examined under epifluorescence microscopy using appropriate filters. For each type of immunocytochemistry and immunofluorescence, control experiments were performed by omission of the primary antibodies, resulting in negative staining.
Quantitative analysis
Three quantitative parameters were evaluated: the size and number of single- and double-labeled endocrine cells, which provide a semiquantitative approach to their secretory activity and the degree of NOS upregulation in different endocrine situations, and the LH plasma levels, a biochemical parameter usually used as an index of gonadotroph activity.
Single (NOS or LH)- and double (NOS and LH)- stained cell profiles were counted in four rats per group to obtain a semiquantitative estimate of the proportion of gonadotrophs showing NOS activity in each experimental group. Counts were performed in five randomly selected sections per rat, dividing each section into square fields of 300 µm x 250 µm at x400. Measurements of the maximal diameter of gonadotrophs (LH-immunopositive cells) were made at x400 by projecting the image with a camera lucida drawing tube onto a calibrated surface (n = 100 cells per rat and group). Only cell profiles including the sectioned nucleus were analyzed. Numerical measurements were corrected by using the assumption-based method of
Statistical analysis
Numerical data are expressed as mean ± SEM. A mean value was obtained from each animal, and four animals were used to calculate the mean and SEM. The statistical significances were assessed by analysis of variance (ANOVA) and Tukeys multiple comparison post-hoc tests for differences among groups .
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results show that, in the pituitary gland of both male and female adult rats, many cells are immunoreactive for nNOS, although their distribution pattern differs from each other.
In males, most nNOS activity was detected in small cells with short processes found between larger immunonegative cells (Fig 1A and Fig 2B). Small nNOS-positive cells were present everywhere in the gland. They were usually grouped in small clusters or forming "ring-like" structures with diameters ranging from 30 to 100 µm, which appeared to surround glandular cysts or follicles (F in Fig 1A and Fig 2A2C). Parallel sections immunostained for S-100 (Fig 2C), an FS cell marker, revealed a labeling pattern similar to that of nNOS. Immunopositive cells were small and often appeared in a "ring-like" arrangement. This suggests that, in male rats, nNOS is mainly expressed in FS cells. nNOS activity was also detected in a smaller number of large round or polygonal cells (Fig 1A, arrow) that, as confirmed by LHnNOS double labeling, were gonadotrophs (Fig 2A and Fig 2B, arrows). The quantitative analysis revealed a cell density of 42.0 ± 3.2 gonadotrophs/field (Fig 3A), with a diameter of 16.3 ± 0.9 µm (Fig 3B), although only a small percentage of them (4.4%; Fig 3A) expressed nNOS.
|
|
|
By contrast, in females, nNOS activity was virtually restricted to large cells (Fig 1B) which, as confirmed by LHnNOS double labeling (Fig 2D and Fig 2E), were gonadotrophs. nNOS-positive "ring-like" structures were not identified, and only a few dispersed small cells showed a faint immunoreaction. Therefore, we can state that there is a sexual dimorphism in the distribution pattern of nNOS in the pituitary gland: In males, nNOS is mainly found in FS cells, whereas in females it is found in endocrine cells. Quantitative study of the number and size of LH cells revealed that they were significantly sparser (22.9 ± 1.6 cells/field; p<0.01; Fig 3A) and smaller (diameter of 12.5 ± 0.1 µm; p<0.01; Fig 3B) in females than in males but, in contrast, most of them (89.7%) expressed nNOS.
Male and female rats were subjected to distinct endocrine manipulations in order to study the effect of different endocrine paradigms on the normal patterns of nNOS activity.
In the pituitary gland of castrated male rats, the distribution pattern of nNOS activity underwent a striking change (Fig 1C). FS cells became immunonegative or faintly stained, and the ring-like nNOS-positive structures were not observed. In contrast, LH cells became larger (23.6 ± 0.7 µm; p<0.001; Fig 3B) and most of them (85.2%; p<0.001) were intensely immunoreactive for nNOS (Fig 1C, Fig 2F, and Fig 2G). So, we can say that after castration, the pattern of nNOS activity turned into a "female-like" pattern, but with a reinforcement of normal female features.
After testosterone replacement, the pituitary gland of castrated rats partially recovered its normal nNOS activity pattern. Endocrine cells became immunonegative or weakly stained (Fig 1E), with a significant decrease in the proportion of both LH cells showing nNOS activity (70.1%; p<0.001; see Fig 3A) and their diameter (18.2 ± 0.3; p<0.001), although FS cells remained nNOS immunonegative, at least during the first 3 days of treatment.
In ovariectomized rats, the pattern of nNOS activity was very similar to that of castrated rats (compare Fig 1C and Fig 1D). There was a reinforcement of the features of normal females, with a significant increase in the number of LH cells/field (53.4 ± 2.5; p<0.001), nNOS cells/field (54.9 ± 0.3; p<0.001), and LHnNOS cells/field (49.6 ± 0.8; see also Fig 2H and Fig 2I), as well as an increase in the size of LH cells (23.1 ± 0.2 µm; p<0.001; Fig 3B). FS cells were nNOS-immunonegative.
In lactating rats, there also was an increase in the number of nNOS-positive cells. The pattern of nNOS activity was similar to that of ovariectomized and castrated rats, with a large number of nNOS-positive cells that correspond in size, shape and distribution to endocrine cells (Fig 1F). Double immunofluorescence for LH and nNOS enabled us to confirm that, in a similar way to what occurs in gonadectomized rats, they were gonadotrophs (Fig 2J and Fig 2K), although in this experimental group they were smaller (14.2 ± 0.2 µm; Fig 3B). Double immunofluorescence for LH and prolactin did not show co-expression.
An additional morphological finding worth mentioning concerns the distribution of endocrine cells. Although present throughout the entire anterior lobe of the pituitary gland, endocrine cells tend to organize in small clusters composed of four to six cells around holes with a diameter of 2040 µm, resembling vascular sinusoids (Fig 2F2I, arrows; see also
Serum LH levels in the different experimental groups are shown in Fig 3C. As expected, basal LH levels in intact male and female rats were low, although slightly higher in females, 6.4 ± 0.0 ng/ml and 8.9 ± 1.4 ng/ml, respectively. Gonadectomy significantly increased these levels in both sexes, 21.8 ± 1.5 ng/ml (p<0.001) in males, and 19.9 ± 1.4 ng/ml (p<0.001) in females. Testosterone treatment significantly reduced castrated levels but not to basal levels (11.5 ± 1.6 ng/ml). Lactating females showed significantly lower LH levels than intact females (3.4 ± 0.4 ng/ml; p<0.017).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Functional studies attribute an important role to NO in the control of gonadotropin secretion and therefore of reproductive functions. Several authors agree that, at the hypothalamic level, NO exerts a positive influence on the biosynthesis and secretion of LHRH (
Confirming previous reports (
FS cells have been defined as a type of non-hormone-secreting cell in the anterior pituitary gland, sharing functional and biochemical characteristics with macrophages (
Several studies have shown that NO has a role in prolactin secretion and lactation. Intracerebroventricular injection of NO donors stimulates prolactin secretion by inhibiting tyrosine hydroxylase activity in the median eminence (
In conclusion, bearing in mind this and previous studies, we propose that NO effects on pituitary hormone (LH and prolactin) secretion depend on the site of release and its cellular source. It exerts a stimulatory effect at a suprapituitary level. At a pituitary level, it also exerts a stimulatory effect when released from LH cells but its effect is inhibitory when released from FS cells.
![]() |
Acknowledgments |
---|
Supported by a grant from Plan Nacional de I+D. Programa Sectorial de Promoción General del Conocimiento, Ministerio de Educación y Cultura (PM95-0060), and by a grant from Gobierno Autónomo de Canarias (PI 1998/008).
Received for publication February 1, 2000; accepted June 28, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abercrombie M (1946) Estimation of nuclear population from microtome sections. Anat Rec 94:239-247
Baes M, Allaerts W, Denef C (1987) Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perfused anterior pituitary cell aggregates. Endocrinology 120:685-691[Abstract]
Bonavera JJ, Sahu A, Kalra PS, Kalra SP (1993) Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: involvement of excitatory amino acids. Endocrinology 133:2481-2487[Abstract]
Bonavera JJ, Sahu A, Kalra PS, Kalra SP (1994) Evidence in support of nitric oxide involvement in the cyclic release of prolactin and LH surges. Brain Res 660:175-179[Medline]
Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768-770[Medline]
Bredt DS, Snyder SH (1992) Nitric oxide, a novel neuronal messenger. Neuron 8:3-11[Medline]
Brunetti L, Preziosi P, Ragazzoni E, Vacca M (1993) Involvement of nitric oxide in basal and interleukin-1ß-induced CRH and ACTH release in vitro. Life Sci 53:PL219-222[Medline]
Brunetti L, Ragazzoni E, Preziosi P, Vacca M (1995) A possible role for nitric oxide but not for prostaglandin E2 in basal and interleukin-1-beta-induced PRL release in vitro. Life Sci 56:PL277-283[Medline]
Catt KJ, Dufau ML (1991) Gonadotropic hormones: biosynthesis, secretion, receptors and actions. In Yen SSC, Jaffe RB, eds. Reproductive Endocrinology. Philadelphia, WB Saunders, 105-155
Ceccatelli S (1997) Expression and plasticity of NO synthase in the neuroendocrine system. Brain Res Bull 44:533-538[Medline]
Ceccatelli S, Hulting AL, Zhang X, Gustafsson L, Vilar M, Hökfelt T (1993) Nitric oxide synthase in the anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc Natl Acad Sci USA 90:11292-11296[Abstract]
Coggeshall RE, Lekan HA (1996) Methods for determining numbers of cells and synapses: a case for more uniform standards of review. J Comp Neurol 364:6-15[Medline]
Denef C (1986) Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab 15:1-32[Medline]
Duvilanski BH, Zambruno C, Seilicovich A, Pisera D, Lasaga M, Diaz MC, Belova N, Rettori V, McCann SM (1995) Role of nitric oxide in control of prolactin release by the adenohypophysis. Proc Natl Acad Sci USA 92:170-174[Abstract]
Garthwaite J, Boulton CL (1995) Nitric oxide signaling in the central nervous system. Annu Rev Physiol 57:683-706[Medline]
González D, Aguilar E (1999) In vitro, nitric oxide (NO) stimulates LH secretion and partially prevents the inhibitory effect of dopamine on PRL release. J Endocrinol Invest 22:772-780[Medline]
González MC, Linares JD, Santos M, Llorente E (1996) Effects of nitric oxide donors sodium nitroprusside and 3-morpholino-sydnonimine on prolactin secretion in conscious rats.. Neurosci Lett 203:167-170[Medline]
González MC, Llorente E (1998) Methylene blue inhibits stimulatory effect of sodium nitroprusside but not of 3-morpholino sydnonimine on prolactin secretion in freely moving male rats.. Brain Res Bull 46:229-231[Medline]
González MC, Llorente E, Abreu P (1998) Sodium nitroprusside inhibits the tyrosine hydroxylase activity of the median eminence in the rat. Neurosci Lett 254:133-136[Medline]
GonzálezHernández T, Pérez de la Cruz MA, MantolánSarmiento B (1996) Histochemical and immunohistochemical detection of neurons that produce nitric oxide: effect of different fixative parameters and immunoreactivity against nonneuronal NOS antisera. J Histochem Cytochem 44:1399-1413[Abstract]
Griffith OV, Stuehr DJ (1995) Nitric Oxide Synthases: properties and catalytic mechanism. Annu Rev Physiol 57:707-736[Medline]
Kato M (1992) Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells. Endocrinology 131:2133-2138[Abstract]
Klatt P, Schmidt K, Brunner F, Mayer B (1994) Inhibitors of brain oxide synthase. Binding kinetics, metabolism and enzyme inactivation. J Biol Chem 269:1674-1680
Kovacs K, Horvath E (1975) Gonadotrophs following removal of the ovaries: a fine structural study of human pituitary glands. Endokrinologie 66:1-8[Medline]
Matsumoto T, Nakane M, Pollock JS, Kuk JE, Förstermann U (1993) A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative. Neurosci Lett 155:61-64[Medline]
Moncada S, Higgs A (1993) The L-arginine-nitric oxide pathway. N Engl J Med 329:2002-2012
Moretto M, Lopez FJ, NegroVilar A (1993) Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 133:2399-2402[Abstract]
Okere CO, Murata E, Higuchi T (1998) Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction. Brain Res 784:337-340[Medline]
Pinilla L, González D, TenaSempere M, Aguilar E (1998) Nitric oxide (NO) stimulates gonadotropin secretion in vitro through a calcium-dependent, cGMP-independent mechanism. Neuroendocrinology 68:180-186[Medline]
Rettori V, Belova N, Dees WL, Nyberg CL, Gimeno M, McCann SM (1993) Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc Natl Acad Sci USA 90:10130-10134[Abstract]
Rivier C (1995) Blockade of nitric oxide formation augments adrenocorticotropin released by blood-borne interleukin-1 beta: role of vasopressin, prostaglandins, and alpha 1-adrenergic receptors. Endocrinology 136:3597-3603[Abstract]
Vanhatalo S, Soinila S (1995) Nitric oxide synthase in the hypothalamo-pituitary pathways. J Chem Neuroanat 8:165-173[Medline]
Vankelecom H, Andries M, Billiau A, Denef C (1992) Evidence that folliculo-stellate cells mediate the inhibitory effect of interferon- on hormone secretion in rata anterior pituitary cell cultures. Endocrinology 139:3537-3546
Vankelecom H, Matthys P, Denef C (1997) Inducible nitric oxide synthase in the anterior pituitary gland: induction by interferon-gamma in a subpopulation of folliculostellate cells and in an unidentifiable population of non-hormone-secreting cells. J Histochem Cytochem 45:847-857
Wang H, Christian HC, Morris JF (1997) Dissociation of nitric oxide synthase immunoreactivity and NADPH-diaphorase enzyme activity in rat pituitary. J Endocrinol 154:R7-11[Abstract]
Wang H, Li S, Pelletier G (1998) Role of nitric oxide in the regulation of gonadotropin-releasing hormone and tyrosine hydroxylase gene expression in the male rat brain. Brain Res 792:66-71[Medline]
Wynick D, Venetikou MS, Critchley R, Burrin JM, Bloom SR (1990) Flow cytometric analysis of functional anterior pituitary cells from female rats. J Endocrinol 126:261-268[Abstract]