Abstract
The expression of the rat quiescin sulfhydryl oxidase (rQSOX) and its putative regulation by estrogens were investigated in the adenohypophysis. Immunohistochemical observations revealed that rQSOX protein is abundantly expressed throughout the anterior lobe of the pituitary, and can be found in almost all the different cell populations. However, as shown by double immunohisto-chemistry, the cells displaying the strongest rQSOX labeling belong to a subset of gonadotrophs. Immunoelectron microscopy showed that, in adenohypophyseal cells, the protein is linked to the membranes of the rough endoplasmic reticulum, the Golgi apparatus and to dense-core secretory granules. These results are consistent with the secretion of the protein and its presumed role in the extracellular matrix. According to its sulfhydryl oxidase function, rQSOX could also participate in the intracellular folding of secreted proteins or hormones like LH and FSH and act as an endogenous redox modulator of hormonal secretion. A semiquantitative RT-PCR analysis of rQSOX level across the estrous cycle and the fact that chronic administration of 17 β-estradiol to ovariectomized rats led to a sustained up-regulation of rQSOX in the pituitary suggest that rQSOX expression is controlled by sex hormone levels. Further investigations are needed in order to elucidate its precise roles in that gland and the mechanisms of its regulation.
Introduction
The rat quiescin sulfhydryl oxidase (rQSOX), a 64 kDa glycoprotein, is a member of the quiescin Q6/FAD (flavin adenine dinucleotide)-dependent sulfhydryl oxidase (QSOX) family (Hoober et al. 1999a, Benayoun et al. 2001, Thorpe et al. 2002). QSOX proteins display a highly conserved structure with an N-terminal PDI (protein disulfide isomerase)-like thioredoxin domain and a C-terminal ERV1 (essential for respiration and vegetative growth)-like domain which contains the FAD-binding site and the redox-active CXXC motif (Coppock et al. 1998). Among them, the most investigated are the human quiescin Q6, whose mRNA level is increased when lung fibroblasts enter reversible quiescence (Coppock et al. 1993, 1998), sulfhydryl oxidases from chicken egg white (Hoober et al. 1996, 1999a, Hoober & Thorpe 1999), guinea-pig endometrial cells (SOx-3, Musard et al. 2001), mouse epidermis (Matsuba et al. 2002) and human neuroblastoma cells (SOXN, Wittke et al. 2003). QSOX proteins catalyze the formation of disulfide bonds in peptides and proteins with reduction of molecular oxygen to hydrogen peroxide (H2O2). They are secreted and are thought to be involved in a broad range of essential cell functions such as elaboration of the extracellular matrix, protein folding, control of the cell cycle and regulation of the redox state through their oxidative effects (Coppock et al. 1998, Hoober & Thorpe 1999, Hoober et al. 1999b, Thorpe et al. 2002).
rQSOX was first identified in the reproductive tract (Chang & Morton 1975) and its mRNA was recently sequenced from seminal vesicles where the protein is highly expressed (Benayoun et al. 2001). It was also detected in a wide range of rat tissues (Benayoun et al. 2001, Mairet-Coello et al. 2002). In a previous paper, we demonstrated that two rQSOX transcripts are expressed throughout the rat brain, the predominant one probably corresponding to the unique transcript detected in peripheral organs (Mairet-Coello et al. 2002). We also mapped the rQSOX protein in the adult rat brain (Mairet-Coello et al. 2004) showing that it is differentially distributed in the central nervous system, notably with a strong expression in neuron populations displaying a high amount of disulfide bond-containing peptides. In the supraoptic nucleus, where its ultrastructural localization was investigated, it is essentially present in the Golgi apparatus and dense-core granules.
An interesting field of research is the study of the estrogen responsiveness of QSOX genes. Effects of the estrogen status on these enzymes are not well documented yet. The SOx-3 and Q6 genes have been shown to be down-regulated by estrogens respectively in endometrial cells (Musard et al. 2001) and MCF-7 breast cancer cells (Inoue et al. 2002); but no investigations have been undertaken concerning estrogen effects on rQSOX expression. Estrogens are important physiological regulators of the secretory activity of the anterior pituitary lobe. In that gland, they exert gene- and cell-specific regulations with positive or negative actions. For example, estrogens stimulate prolactin (PRL) synthesis and secretion in the lactotrophs (Perez et al. 1986, Shull et al. 1987, Maeda et al. 1996) and regulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion in the gonadotrophs (Shupnik 1996, McNeilly et al. 2003). While mapping rQSOX distribution in rat brain, our attention was attracted by an intense labeling of many adenohypophyseal cells. In the present work, we thus investigated rQSOX expression in the adult rat pituitary using RT-PCR and immunohistochemistry; we determined its subcellular localization and characterized pituitary cell populations expressing it by double-immunohistochemical procedures. Finally, a semiquantitative RT-PCR protocol was used to analyze the putative changes of rQSOX mRNA levels in pituitaries of adult female rats throughout the estrous cycle as well as in ovariectomized rats treated or not with 17 β-estradiol.
Materials and Methods
Animals and experimental protocols
Adult male and female 250–300 g Sprague–Dawley rats (IFFA Credo, L’Arbresle, France) were housed in a temperature-controlled environment under natural light conditions with food and water available ad libitum. All animal manipulations and experimental protocols were performed according to the recommendations of our institution and under the supervision of authorized investigators.
Estrous cycle experiments
The estrous cyclicity was monitored by daily cytological examination of vaginal smears between 1000 h and 1200 h. Only rats exhibiting at least two consecutive 4-day estrous cycles were selected for the study. Animals were killed by decapitation at 1400 h on each day of the estrous cycle: proestrus (P), estrus (E), metestrus (M) and diestrus (D) (five animals were used for each data point of the cycle). Pituitary glands were rapidly removed, frozen over liquid nitrogen and stored at −80 °C until RNA isolation.
Ovariectomy and estrogen administration
Twelve adult female rats were bilaterally ovariectomized (OVX) under ketamine/xylazine anesthesia. After a 3-week recovery period, OVX rats were divided into two groups of six rats. The first group (OVX+E2, n=6) received a daily estrogen injection (β-estradiol 3-benzoate, Sigma; 25 μg/kg body weight, diluted in 5% ethanol, s.c.) for 9 days. The second group (OVX, n=6) received the ethanol vehicle injection (equivalent volume, s.c.) for the same period. The rats were killed by decapitation 4 h after the last injection and the pituitary glands were collected, frozen over liquid nitrogen and stored at −80 °C until RNA preparation. The experiment was repeated twice. To assess the effects of estradiol chronic administration, all rats were weighed before the first and after the last injection; then, body weights of the treated group were compared with those of the control group. In addition, at the end of the experiments, uteruses were isolated and macroscopically examined to compare their size in treated animals and controls.
Light microscope immunohistochemistry
Four male and four female rats were deeply anesthetized with 7% chloral hydrate solution (5 ml/kg body weight). After transcardial perfusion of the animals with 300 ml 0.9% NaCl followed by 300 ml of fixative (ice-cold 4% paraformaldehyde solution in 0.1 M phosphate buffer (PB), pH 7.2), pituitaries were removed, post-fixed in the same fixative for 2 h at 4 °C, and immersed overnight in a 15% cryoprotective sucrose solution in 0.1 M PB at 4 °C. They were embedded in a commercial medium (Cryomatrix, Shandon, Pittsburgh, PA, USA), quickly frozen over liquid nitrogen, then serially cut at a thickness of 10 μm with a cryostat-microtome; the sections were mounted on gelatinized glass-slides and stored at −45 °C until treatment.
For rQSOX detection, after rinsing in 0.1 M PBS (pH 7.2), containing 0.3% triton X-100 (PBS-T), sections were submitted to a classical indirect immunofluorescence protocol. As previously described (Mairet-Coello et al. 2004), sections were incubated overnight with the rabbit rQSOX polyclonal antiserum prepared by Benayoun et al.(2001) and diluted 1/500 in a PBS-T solution containing 10% lactoproteins, 1% BSA and 0.01% sodium azide. After washing in PBS-T, sections were then exposed for 1 h to secondary goat anti-rabbit Alexa Fluor 488-conjugated immunoglobulin G (Molecular Probes, Interchim, Montluçon, France; diluted 1/400 in the same solution as the primary). The rQSOX antiserum we used revealed only one protein as attested by Western blots on seminal vesicle fluid and whole brain homogenates (Benayoun et al. 2001, Mairet-Coello et al. 2004). In the present study, specificity of the stainings was also checked by incubating the rQSOX antiserum (1/500) for 4 h at room temperature with the antigen solution (0.03 nmol of rQSOX protein purified from seminal vesicle fluid per microliter of non-diluted antiserum) prior to using it for immunostaining. Sections were observed under a fluorescence microscope (Olympus BX51).
In order to characterize which anterior pituitary cell populations express rQSOX, double- immunohistochemical procedures were performed either on cryostat or on semi-thin sections by combining peroxidase anti-peroxidase (PAP) and indirect immunofluorescence methods. Cryostat sections were first incubated overnight with the rQSOX antiserum and revealed through the PAP (DAKO, Trappes, France) procedure using 3,3′-diaminobenzidine (DAB), then treated with the second primary (anti-LH (1/200), anti-FSH (1/200), anti-growth hormone (GH, 1/200), anti-PRL (1/200), anti-β-endorphin (1/200) from Biogenex Laboratories, San Ramon, CA, USA; and anti-thyroid stimulating hormone (TSH, 1/50) from DAKO) which was revealed using a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (1/400) or a fluorescein isothiocyanate (FITC)-labeled anti-mouse antibody (1/100, Jackson Immunoresearch Laboratories Inc., Interchim, Montluçon, France) for the monoclonal anti-TSH antibody. Semi-thin sections were obtained from thick sections treated with the rQSOX antiserum and revealed through the PAP procedure before embedding (see below). They were incubated overnight with the second primary and revealed as described above.
Electron microscope immunohistochemistry
The technique was adapted from Li et al.(2000). Briefly, male rats anesthetized with 7% chloral hydrate solution were perfused at room temperature with 0.1 M PB containing 4% paraformaldehyde, 15% saturated picric acid (v/v) and 0.05% glutaraldehyde. Pituitary glands were quickly removed. The anterior and the posterior lobes of the pituitaries were separated, placed in 0.1 M PB and immersed for 30 min in 0.05 M PB containing 25% sucrose and 10% glycerol (v/v) for cryoprotection. Anterior pituitaries were then quickly frozen over liquid nitrogen and cut into serial sections (40 μm thick) on a slicer microtome. Slices were immersed for 1 h in 0.05 M Tris-buffered saline (pH 7.4) containing 20% normal goat serum. After a 60 h incubation at 4 °C with the rQSOX antiserum (1/500 in 0.05 M Tris-buffered saline, pH 7.4, containing 2% goat serum), they were exposed for 24 h to goat anti-rabbit immunoglobulin G (1/100, P.A.R.I.S., Compiègne, France) followed by a 24 h incubation with PAP (1/200). Peroxidase was revealed with 0.02% DAB in 0.05 M Tris–HCl containing 0.003% H2O2. Slices were then embedded in araldite and cut into 1 μm sections for double immunohistochemistry. For the electron microscope study, slices were incubated for 1 h in 1% osmium tetroxide in 0.1 M PBS, then embedded and sectioned in ultrathin sections before being examined on a Jeol 1230 electron microscope.
Photomicrograph production
For light microscopy, digital image acquisitions were carried out using a DP50 Olympus camera and the AnalySIS 3.1 software (Soft Imaging System). Electron micrographs were also numerized through the camera associated with the Jeol microscope using AnalySIS software. Images were imported in Adobe Photoshop 7.0 software for treatment. Transformation of color images into black and white images was performed using gray-scale functions minimally altering the captured images.
Total RNA extraction
Total RNAs were extracted from individual pituitaries using an RNA extraction kit (RNA NOW, Ozyme, Saint Quentin en Yvelines, France) according to the manufacturer’s instructions and as previously described (Mairet-Coello et al. 2002). RNA concentrations were determined by 260 nm absorbance using a spectrophotometer (Eppendorf, Le Pecq, France).
RT-PCR and semiquantitative RT-PCR
Denatured total RNAs (1 μg) were reverse transcribed into cDNAs in a 20 μl reaction mixture consisting of 4 μl of 5 × reverse transcriptase reaction buffer (Promega), 5 μM random hexamer primers (Amersham), 1.25 mM mixture of the four deoxyribonucleotides (Roche), 20 U RNasin® (Promega) and 100 U Moloney murine leukemia virus reverse transcriptase (Promega). RT reactions were incubated for 10 min at 23 °C, 1 h at 42 °C, 5 min at 95 °C and cooled to 4 °C in a PTC-200 thermocycler (MJ-Research, Fontenaysous-Bois, France).
PCRs were performed to amplify a 797 bp fragment of rQSOX cDNA (base 17 to base 813, GenBank accession number NM_053431) using the fluorescent rQSOX sense primer 5′-6-FAM-ACTTGAGCGAGGTGGACAGTC AAG-3′ and the rQSOX antisense primer 5′-AGCACA GGCACTCGGGAA-3′ (Eurogentec, Seraing, Belgique; Benayoun et al. 2001, Mairet-Coello et al. 2002). For quantitative experiments, additional primers amplifying a 509 bp fragment of cyclophilin cDNA (CYC, GenBank accession number M19533), a housekeeping gene, were included in the PCR mixture as co-amplified internal control for normalization (CYC sense primer 5′-CGCC GCTTGCTGCAGACATGG-3′ and fluorescent CYC antisense primer 5′-6-FAM-GAGTTGTCCACAGTCG GAGATGG-3′). All PCR amplifications were carried out in the PTC-200 thermocycler using the FastStart Taq DNA polymerase kit (Roche) in a 20 μl final reaction mixture. Each PCR reaction contained 0.8 μl of the RT product, 2 μl of 10 × PCR buffer, 3.75 mM MgCl2, 0.5 mM mixture of the four deoxyribonucleotides, 1 U Taq DNA polymerase, and rQSOX and CYC primers at optimal concentrations (see Results). Conditions of amplification were established simultaneously with the two sets of primers, by varying the primer dilutions, the cDNA matrix concentration and the number of cycles. Optimal cycle number was determined over a range of 21–35 cycles and was chosen at the beginning of the exponential phase of amplification. Thermocycling parameters were as follows: one step of PCR enzyme activation at 95 °C for 5 min followed by 27–30 cycles (see Results) of denaturation at 95 °C for 30 s, annealing at 63 °C for 30 s and extension at 72 °C for 1 min 30 s. The first investigations to know whether rQSOX is expressed in the rat pituitary involved a standard 30 cycle PCR using a single set of non-fluorescent rQSOX primers (1.5 μM for each primer). For the qualitative detection of rQSOX in the pituitary, 18 μl of rQSOX-PCR product were separated by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized under u.v. illumination using Gel Doc 2000 (BioRad) and the BioRad Quantity One 4·2·3 software. For the quantitative part of the protocol, each sample was denatured by heating for 2 min at 95 °C and snap-cooled before loading. Fluorescent PCR products (1 μl) were separated on a 24 cm 6% polyacrylamide denaturing gel for 6 h at 1200 V on a 373 A DNA sequencer (Applied Biosystems, Courtaboeuf, France). In each electrophoresis lane, intensities of cDNA fluorescent fragments, corresponding to rQSOX (797 bp) and CYC (509 bp) were detected at an absorbance of 500 nm. Electrophoretic data were automatically analysed and sized in base pairs by GENESCAN Analysis software and reconstructed as a gel image.
Statistical analyses
Although internal control compensates for the inherent inter-assay variability of RT-PCR reactions, two RTs were performed for each sample followed by two PCRs for the estrous cycle study and one for the estrogen replacement experiment. Results used for statistics correspond to the average obtained for each animal. rQSOX fluorescence intensity was normalized to CYC fluorescence. All data are presented as percentages of the controls. The controls (100%) correspond to the value at metestrus for estrous cycle experiments and to the value of vehicle-treated animals for estrogen injection experiments. Statistical analyses were performed using the GraphPad InStat 3.05 software. Statistical significance was evaluated by unpaired one-way ANOVA test for multiple groups and by unpaired parametric Student’s t-test for comparisons between two groups. Differences were considered statistically significant when P < 0.05.
Results
Detection of QSOX expression in rat pituitary
Using RT-PCR analysis, an rQSOX fragment of the 797 bp expected size was reproducibly observed in pituitary homogenates from male rats, gonadally intact female rats and ovariectomized vehicle-(OVX) or E2 (OVX+E2)-treated rats (Fig. 1). The amplified fragment corresponds to a part of the 5′ region common to the two transcripts previously identified in the brain (Mairet-Coello et al. 2002).
Light and electron immunocytochemical observations
On cryostat sections, numerous cells were more or less intensely labeled by the rQSOX antiserum throughout the anterior pituitary. The labeling was located in the cytoplasm, being either concentrated at one pole of the cell or staining the whole cytoplasm and exhibiting a more or less granular aspect (Fig. 2A). Preincubating the antiserum with the corresponding antigen completely abolished the immunolabeling (Fig. 2B and C). At the ultrastructural level, rQSOX immunoreactivity was associated with the rough endoplasmic reticulum and golgian compartments; more precisely, a dark labeling underlined the membranes of the rough endoplasmic reticulum cisternae (Fig. 2D) and saccules of the Golgi apparatus (Fig. 2E). A deposit was also noticed either in the dense core (Fig. 2F) or sometimes along the membrane (Fig. 2D) of secretory granules dispersed among large amounts of unlabeled ones. Double immunostainings with antisera to the main pituitary hormones did not allow clear definition of the nature of all rQSOX-positive cells. Only some cells detected by LH and FSH antiserums undoubtedly showed a double labeling. In order to clarify these observations, antisera to the different pituitary hormones were applied to semi-thin sections obtained after an rQSOX-PAP detection performed at a pre-embedding step (see Materials and Methods). Results confirmed that cells showing the strongest rQSOX labeling were those recognized by LH (Fig. 3A and B) and FSH antisera (Fig. 3C and D). Few GH (Fig. 3E and F) and TSH (Fig. 3G and H) cells were lightly stained by the rQSOX antiserum. Very few lactotrophs and corticotrophs were slightly labeled.
Study of a putative effect of estrogens on QSOX expression in rat adenohypophysis
During the estrous cycle
It is well established that estrogen plasma concentrations undergo large variations during the estrous cycle. Thus, we examined the effect of these physiological variations on rQSOX mRNA expression in intact cycling rats. The optimization of semiquantitative RT-PCR conditions (see Materials and Methods) was carried out on pituitaries from rats killed at the metestrus stage. Primers were used at 187.5 nM for each rQSOX primer and 37.5 nM for each CYC primer and PCR amplifications were performed for 27 cycles. The amounts of rQSOX mRNA in the pituitary showed moderate changes during the course of the estrous cycle (Fig. 4). Relative rQSOX mRNA levels (normalized to the housekeeping gene cyclophilin) were highest during estrus and then declined to reach the lowest levels at the proestrus stage (35% decrease compared with the estrus stage).
After ovariectomy and chronic 17 β-estradiol administration
The responsiveness of the animals to the treatment was verified by comparison of body weights between the control (OVX) and the estradiol-treated (OVX+E2) groups before the first and after the last vehicle or estradiol injections. Before treatment, the average weights of the two groups of ovariectomized rats were not significantly different (control group, 344.6±11.19 g; estradiol-treated group, 327.2±6.91 g; P>0.1). Conversely, after the ninth injection, the vehicle-treated ovariectomized rats (349.6±11.61 g) were significantly heavier than those that had received estradiol (306.5±6.39 g, P<0.005). Between the beginning and the end of the treatment, the estradiol-treated group lost weight (−20.7±1.71 g) whereas the control group gained weight (+5±2.1 g, P<0.0001). Moreover, macroscopic examination showed that uteruses of the estradiol-treated rats were larger than those of the controls. As for the amounts of pituitary rQSOX mRNA, optimal conditions of PCR amplifications were established for vehicle-treated rat pituitaries. Each rQSOX primer was used at 1.5 μM, each CYC primer was diluted at 8.3 nM and PCRs were performed for 30 cycles. The treatment of OVX rats with E2 caused a significant average increase of 56% in the ratio of rQSOX mRNA/CYC mRNA compared with the OVX control group (P=0.0069) (Fig. 5A and B).
Discussion
Expression of rQSOX in the anterior pituitary
Previous studies showed that rQSOX is widely distributed throughout the rat brain (Mairet-Coello et al. 2002, 2004) and is present in most of the peripheral organs with a high amount in the male reproductive tract (Benayoun et al. 2001, Mairet-Coello et al. 2002). Despite this ubiquitous expression, few studies have been devoted to sulfhydryl oxidases. The present data demonstrate the occurrence of rQSOX in rat pituitary. It is expressed both in male and female animals and the protein is abundant throughout the anterior lobe. The subcellular localization of rQSOX in the adenohypophysis is in agreement with our previous data showing the association of the protein with the Golgi apparatus and the dense-core granules in neurons of the supraoptic nucleus (Mairet-Coello et al. 2004). In anterior pituitary cells, the protein was also found to be linked to the membranes of the ergastoplasmic compartment, as was shown for human QSOX (Thorpe et al. 2002). These results suggest that QSOX proteins, which possess a putative signal peptide, can follow the secretory pathway and probably play a role in the extracellular space, as previously proposed (Coppock et al. 1993, Hoober & Thorpe 1999, Hoober et al. 1999b, Benayoun et al. 2001, Thorpe et al. 2002, Mairet-Coello et al. 2004).
The strong expression of rQSOX in a secretory gland such as the hypophysis is not surprising. Indeed, sulfhydryl oxidases are enzymes that catalyze the formation of disulfide bonds (Hoober et al. 1996, Benayoun et al. 2001) and most eukaryote secreted proteins contain disulfide bridges (Wittrup 1995). Disulfide bonds and cysteine thiols could play a decisive role in protein folding and secretion; their manipulation affects intracellular transport and release (Wittrup 1995, Van Horssen et al. 1998, Gorr et al. 1999). QSOX proteins have been shown to be abundantly expressed in secretory tissues producing disulfide-containing peptides and proteins such as skin apocrine glands, parotid glands, pancreas and small intestine (Thorpe et al. 2002); in addition, we recently reported an intense rQSOX immunoreactivity in neurons synthesizing neuropeptides or neurohormones which possess disulfide bridges such as melanin-concentrating hormone, hypocretin/orexin, vasopressin and ocytocin (Mairet-Coello et al. 2004). Although rQSOX immunoreactivity was more or less detected in most of the hormone-secreting cell types of the adenohypophysis (gonadotrophs, somatotrophs, lactotrophs and thyrotrophs), double-immunolabeling experiments provided evidence that the anterior pituitary cells strongly expressing rQSOX correspond to a subset of gonadotrophs. However, more detailed analyses are needed to clarify whether rQSOX-positive cells belong to the subpopulation of gonadotrophs which expresses both LH and FSH and/or to mono-hormonal subsets (Kovacs & Horvath 1985). LH and FSH are members of the glycoprotein hormone family which regroups non-covalently bounded heterodimers made up of the assembly of a common α-subunit and a distinct β-subunit that confers biological specificity. Interestingly, each subunit contains several cysteine amino acid residues that are involved in multiple intramolecular disulfide linkages which are required to allow subunit interactions to form the biologically active dimers (Reeve & Pierce 1981, Chin 1985, Muyan et al. 1998). Thus, in addition to its action on extracellular components, rQSOX could also participate intracellularly in different steps of oxidizing folding of secreted proteins, from the initial formation of disulfide linkages that occurs in the rough endoplasmic reticulum until the maturation and the maintenance of the disulfide-bonded protein structure in the golgian compartment and the secretory vesicles. It still remains to be verified whether rQSOX and disulfide-containing hormones like LH and FSH are colocalized in the same compartments and whether they are co-secreted. Although TSH, GH and PRL contain intra-chain disulfide bridges (Reeve & Pierce 1981, Chin 1985, Seo 1985), rQSOX was slightly expressed by thyrotrophs, somatotrophs and lactotrophs. This means that rQSOX preferentially exerts its oxidizing activities on LH and FSH while the other hormones probably use other sulfhydryl oxidases or mechanisms of disulfide bond production.
Interestingly, an intense rQSOX labeling was observed in neurosecretory neurons of the hypothalamus that control the adenohypophyseal secretions – such as the parvicellular cells of the anterior periventricular, median preoptic, paraventricular and arcuate nuclei (Mairet-Coello et al. 2004). The cellular redox state might regulate secretion and action of hormones; for example, nitric oxide and thioredoxin, two redox-modulatory substances, could be implicated in the hypothalamic–pituitary functions, notably in the modulation of GH (Kato 1992, Hata et al. 2001) and LH secretions (McCann et al. 1996, 2003); cellular glucocorticoid hormone responsiveness is negatively modulated by antisense thioredoxin expression and treatment with H2O2 (Makino et al. 1996). A rapidly increasing body of evidence indicates that reactive oxygen species, in particular H2O2, could play an important role as signaling molecules involved, for example, in cellular proliferation or activation, growth inhibition or cell death (Nakamura et al. 1997, Gabbita et al. 2000, Hensley et al. 2000). Through the production of H2O2, rQSOX could thus act as an endogenous cellular redox modulator in the hypothalamic–pituitary axis.
Effect of estrogens on rQSOX expression in the pituitary
Our experimental data, together with the variations of rQSOX expression during the estrous cycle, suggest that rQSOX expression is related to the estrogen level. The quantitative experiments were conducted on homogenates from whole pituitaries and so, they show global variations of rQSOX expression. However, it cannot be excluded that the variations could be restricted to specific cell populations of the pituitary. rQSOX immunoreactivity (see above) was present in almost all of the adenohypophysis cell types but was predominant in the gonadotrophs which constitute 5–10% of the total anterior pituitary cells (Dada et al. 1983). If the changes of its expression are restricted to this small population (or another), it could partially explain the moderate variations detected across the estrous cycle and the lack of statistical significance. Moreover, two rQSOX transcripts, probably arising from an alternative splicing, have been identified in rat brain by Northern blot, but only the short transcript was revealed in several peripheral organs (Mairet-Coello et al. 2002). A sulfhydryl oxidase activity has been shown for the protein encoded by the short mRNA (Benayoun et al. 2001), but no investigations have been undertaken yet for the putative protein encoded by the long transcript. The primers we used for the semiquantitative PCR analysis are located in the 5′ part of the cDNA sequence common to the two presumed transcripts and this did not enable us to distinguish them by PCR. We do not know yet whether the second transcript is also expressed in the pituitary; if this is the case, the quantifications realized could correspond to the expression of both mRNAs; however, it is also possible that they are differentially regulated by estrogens. The existence of such a differential regulation has been previously reported in the literature; for example, several estrogen receptor mRNA isoforms were specifically modulated in rat pituitaries throughout the estrous cycle as well as in response to steroid hormones (Friend et al. 1995, 1997). Further experiments are thus necessary to determine whether both rQSOX splice variants are present in the pituitary and, if that is the case, to analyze each of them separately.
In our conditions, higher rQSOX expression was seen at estrus and the minimum expression at proestrus, suggesting that it may be linked to the levels of physiological sex steroids. In the rat, the estrogen peak is reached in the late afternoon of the proestrus stage, approximately 8 h before the beginning of estrus (El Meskini et al. 1997). This implies either that rQSOX responds to the elevation of the estrogen level with a delay, or that the peak of rQSOX expression happens during a period of the estrous cycle that we did not examine, i.e. between the afternoon of proestrus and the early afternoon of estrus. Then, rQSOX expression declines slowly after the estrogen level falls to reach a minimum at the proestrus stage just before the following peak of estrogens. The administration of 17 β-estradiol to ovariectomized rats showed that rQSOX expression is up-regulated by estrogens. But we cannot exclude an influence of progesterone impregnation as well. The guinea-pig sulfhydryl oxidase SOx-3 was discovered by looking for estrogen-regulated genes and its levels of expression in the endometrium changed across the estrous cycle. Conversely to rQSOX in rat pituitary, SOx-3 expression in guinea-pig endometrium was lowered during the estrus phase and was supposed to be down-regulated by estrogens (Musard et al. 2001). Moreover, a screening of estrogen genomic effects has recently demonstrated that the human quiescin Q6 gene is repressed by estrogens in MCF-7 breast cancer cells (Inoue et al. 2002). These data suggest species- and/or tissue-specific modulations of QSOX gene expression. Tissue-dependent regulation by estrogens has been previously described for several other genes such as vasoactive intestinal peptide (Kasper et al. 1992), angiotensinogen (Gordon et al. 1992) and gastrin-releasing peptide (Whitley et al. 2000).
The apparent delayed response during the estrous cycle and the sustained increase of rQSOX level in pituitary after chronic administration of estrogens raise the possibility of indirect estrogenic effects. Indeed, estrogens are known to affect pituitary function either directly or indirectly via the hypothalamus. The majority of gonadotrophs do not express estrogen receptors (Mitchner et al. 1998) and there is a general consensus that estrogens affect the gonadotrophs mainly via the hypothalamus (McCann et al. 1998). For example, it is well established that estrogens regulate the reproductive hormones LH and FSH by primarily altering the release of gonadotropin-releasing hormone (GnRH) (Shupnik 1996). In the same way, estrogens could affect rQSOX expression in pituitary indirectly through the hypothalamic gonadoliberins.
In conclusion, we report the occurrence of QSOX throughout the anterior lobe of rat pituitary, mainly in gonadotrophs. Associated with intracellular compartments belonging to the secretory pathway, this enzyme may modulate secretory activity by participating in the folding of hormones containing disulfide bonds and by regulating the redox state. We also show that rQSOX expression is correlated to estrogen levels. However, further investigations are needed to elucidate the mechanisms of its regulation and its precise role in the pituitary.
RT-PCR detection of rQSOX. RT-PCR were performed on 1 μg of total RNAs extracted from each sample, using specific rQSOX primers. After agarose gel electrophoresis and ethidium bromide staining, a band of 797 bp was detected in pituitaries of male (two separate individuals), cycling female (P, proestrus; E, estrus; M, metestrus; D, diestrus) and untreated (OVX) or 17 β-estradiol (OVX+E2)-treated ovariectomized rats.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05842
Immunolocalization of rQSOX. (A, B and C) Light microscope photographs: (A) in adenohypophyseal cells, the rQSOX immunolabeling is located in the whole cytoplasm (arrowhead) or concentrated at one pole of the cell (arrows); (B and C) preincubation of the rQSOX antiserum with the antigen completely abolished the labeling (C) observed in the precedent section (B). (D, E and F) Electron microscope photographs: (D) the labeling is associated with the membranes of ergastoplasmic cisternae (arrows) and the membranes of some secretory granules (arrowheads); (E) labeled membranes of saccules of the Golgi apparatus cut into longitudinal (arrows) or transversal (arrowheads) planes; (F) immunoreactive dense-core granules (arrows) among unlabeled ones (arrowheads). Scale bars: 50 μm (A, B and C); 500 nm (D, E and F).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05842
Double immunohistochemistry for rQSOX (PAP) and the main anterior pituitary hormones (immunofluorescence). (A and B) Numerous LH-positive cells (B) are also positive for rQSOX (A). (C and D) Most of the FSH gonadotrophs (D) are also labeled for rQSOX (C). (E and F) Some somatotrophs (F) are slighly stained by the rQSOX antiserum (E). (G and H) Few cells are double labeled for TSH (H) and rQSOX (G). Scale bars: 50 μm.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05842
Variations of QSOX mRNA levels in pituitary glands of female rats across the estrous cycle as determined by semiquantitative RT-PCR. Pituitaries were collected at 1400 h on proestrus (P), estrus (E), metestrus (M) and diestrus (D) stages. Total RNAs (1 μg) from individual pituitaries were reverse transcribed and relative quantitative PCR analyses were performed using fluorescent primers. The intensity of the fluorescent products was quantified on a 373 A DNA sequencer and analysed with the GENESCAN Analysis software. rQSOX cDNA level was normalized to that of the coamplified internal control cDNA signal (cylophilin, CYC). For each stage, data from five rats are shown as means±s.e.m. of two independent RTs followed by two PCRs per RT. Results are expressed as a percentage after setting the level in the metestrus stage at 100%.
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05842
Effects of 17 β-estradiol replacement on QSOX mRNA content in pituitaries of ovariectomized rats. After ovariectomy, animals received, for 9 days, a daily s.c. injection of vehicle (OVX) or estradiol benzoate (25 μg/kg per day, OVX+E2). Total RNAs were prepared from each pituitary and analysed by semiquantitative RT-PCR as indicated in Fig. 4. (A) Representative fluorescent PCR signals from OVX and OVX+E2 rats on polyacrylamide gel reconstructed by GENESCAN software. (B) Bars represent the ratio of rQSOX cDNA levels normalized to the cyclophilin (CYC) signal. Data are shown as means±s.e.m. of two independent series, each containing six control rats (OVX) and six rats treated by estradiol (OVX+E2); two independent RT-PCR were assayed for each sample. Results are expressed as a percentage after setting the level in the vehicle control at 100%. The asterisk indicates that the values are significantly different between the two groups, using Student’s t-test (*P=0.0069).
Citation: Journal of Endocrinology 183, 2; 10.1677/joe.1.05842
(A Tury and G Mairet-Coello contributed equally to this work)
The authors thank Dr F Esnard and Dr A Esnard-Fève (INSERM EMI-U-0010, Université François Rabelais, Tours, France) for providing the rQSOX antiserum and Dr A Gouget (Service de Cytogénétique, Centre Hospitalier Universitaire, Besançon, France) for her expert technical advice.
Funding
This work was supported by the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche. There are no conflicts of interest in this study.
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