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Department of Cell Biology and Anatomy I (T.W., Y.O., S.W., Y.U.), Osaka University Medical School, 565 Osaka; and the Doctoral Program in Medical Sciences (T.B.), University of Tsukuba, 305 Tsukuba, Japan; and the Department of Anatomy I (T.J., D.G.), Hannover Medical School, D-30625 Hannover, Germany
Address all correspondence and requests for reprints to: Dr. Tsuyoshi Watanabe, Department of Cell Biology and Anatomy I, Osaka University Medical School, Yamadaoka 2–2, Suita-shi, 565 Osaka, Japan. E-mail: tyshwata{at}anat1.med.osaka-u.ac.jp
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Introduction |
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Although the physiological roles of granins are not fully understood, extensive immunocytochemical investigations have provided some information on their characteristic tissue distributions. Granins are found in various peptide-producing endocrine tissues including the adrenal medulla, endocrine pancreas, parathyroid gland, and pituitary gland, but not in exocrine or epithelial cells (for reviews, see Refs. 3 and 4). Endocrine cells of the mammalian anterior pituitary glands are known to be one of the major storage sites of granins as well as adrenal chromaffin cells. Three representative members of the granin protein family, chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII), have been shown to be densely distributed in gonadotropes among pituitary endocrine cells (5, 6, 7).
The gonadotropins secreted by gonadotropes, LH and FSH, are highly regulated by related hormones or factors such as LHRH, sex steroids, inhibin, and activin (8). Granins are colocalized with gonadotropins in secretory granules of gonadotropes, and are concomitantly secreted in response to stimulation by LHRH (9, 10, 11).
Typical gonadotropes in the male rat pituitary exhibit two distinct subsets of secretory granules. In prior studies, we have shown, using immunocytochemical techniques, that CgA and SgII are localized separately in these morphologically different secretory granules within the gonadotropes: CgA is localized only in large granules, whereas SgII is confined to small granules (12, 13, 14). These different localizations of CgA and SgII indicate that gonadotropes are able to use two different subsets of secretory granules for the purposes of maintaining two distinct regulated secretory pathways, at least for CgA and SgII. However, how these different secretory granules are formed and maintained remains largely unexplained.
The proportions of these two subsets of secretory granules within gonadotropes are not definite but vary depending on the functional states of gonadotropes (15, 16). In addition, sex-related differences exist in the ultrastructure of typical male and female gonadotropes; large secretory granules, which are regularly present in male rat gonadotropes, are obviously very rare in the majority of female gonadotropes (16, 17, 18).
This paper addresses the issue of whether the formation of secretory granules in gonadotropes is affected by sex steroids, which are known to be major gonadal factors and regulate the functional state of gonadotropes. We analyzed the effects of two sex steroids, testosterone and estradiol, on the ultrastructural and biochemical properties of secretory granules in castrated male rat gonadotropes. The results suggest that the expression levels of granins in gonadotropes are regulated differently by male and female sex steroids, and that the appearance of large secretory granules containing CgA in gonadotropes differ depending on the expression levels of CgA in the pituitary.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and experimental procedures
Sixty-four adult male Wistar rats (body weight, 
250 g) were
divided into 4 experimental groups (16 rats each) and kept in plastic
cages placed in a well ventilated room (temperature, 23 ± 1 C;
relative humidity, 55–65%) with food and water ad
libitum.
At 8 weeks of age, three groups (groups B, C, and D) were bilaterally gonadectomized through a scrotal incision under pentobarbital anesthesia, whereas the rats in group A received only sham operation and were used as nontreated control animals. The rats in groups C and D received a sc SILASTIC brand implant (Dow Corning, Midland, MI) containing testosterone (for group C) or ß-estradiol (for group D) according to the method of Goodman (22). The implants, consisting of 15-mm long pieces of SILASTIC medical grade tubing (id, 0.062 in.; od, 0.125 in.; Dow Corning), were filled with 120 mg crystalline testosterone (Wako Pure Chemicals, Osaka, Japan) or ß-estradiol (Sigma Chemical Co., St. Louis, MO). These rats were then maintained for an additional 8 weeks under the same conditions and were then used for experiments as described below.
Tissue and blood plasma preparation for biochemical analyses
After rats were anesthetized with pentobarbital (25 mg/kg, ip),
blood was collected into tubes containing heparin (100 U/tube) by
puncture of the abdominal aorta (n = 5/experimental group). The
blood samples were then centrifuged at 4 C for 10 min, and the plasma
of each sample was divided into aliquots and stored at -20 C until
assay.
For immunoblotting, pituitaries and adrenals were excised from the same animals immediately after blood sampling. Tissue samples were then quickly frozen in liquid nitrogen and stored at -70 C until used.
RIA
Plasma LH concentrations were assayed using a commercially
available RIA kit [rat (rLH) [125I] assay system; code
RPA 552; Amersham International, Aylesbury, UK] according to kit
instructions. Briefly, 100 µl of plasma samples were mixed with 100
µl rabbit antirat LH serum and 100 µl [125I]rat LH
(13,000 cpm/assay tube), and incubated for 16 h at room
temperature. All reagents and samples were dissolved and diluted with
the assay buffer (0.025 M phosphate buffer, pH 7.5,
containing 0.1% sodium azide) included in the kit. Free and bound
fractions of radiolabeled LH were separated by the addition of 400 µl
Amerlex-M second antibody reagent (Amersham), and after centrifugation
for 10 min at 1,500 x g, the radioactivity in the
precipitate was measured in a 
-scintillation counter. The amount of
LH in the sample was read from a standard curve and expressed as
nanograms of the standard preparation of rat LH provided by the
supplier. The sensitivity of the assay, defined as the amount of rat LH
required to reduce the binding of iodinated LH to 95% of zero dose
binding, was 0.08 ng/tube. Statistical analyses were performed by ANOVA
using Tukey’s comparison test.
Immunoblotting
The pituitaries of the experimental animals (n = 5/group)
were individually homogenized in 1 ml 0.05 M Tris-HCl
buffer (pH 7.0), containing 0.15 M NaCl, 1% Triton X-100,
1 mM EDTA, 10 µg/ml leupeptin (Peptide Institute, Osaka,
Japan), 10 µg/ml pepstatin (Peptide Institute), and 0.05
mM p-amidino-phenylmethanesulfonylfluoride
hydrochloride (Wako Pure Chemicals) in a Polytron homogenizer
(Kinematica, Littau, Switzerland) at 80% of the maximal speed for 10
sec. Immediately after homogenization, samples were centrifuged at
10,500 x g for 20 min at 4 C, and the supernatants
were used for immunoblotting.
The pituitary and adrenal (used as controls) extracts were analyzed by 10% SDS-PAGE under reducing conditions. Electrophoretic transfer of proteins from the polyacrylamide gel to Hydrophobic Durapore sheets (Immobilon-P, Millipore, Tokyo, Japan) was performed according to the method of Towbin et al. (23). The sheets were soaked in PBS containing 5% BSA (Sigma Chemical Co.) to block nonspecific binding and then incubated with antisera directed against rat CgA or SgII (diluted 1:200). Immunodetection was carried out with a chemiluminescent ECL kit (Amersham) according to the manufacturer’s recommended protocol.
Preparation of digoxigenin-labeled RNA probes
A complementary DNA (cDNA) fragment covering the entire
open reading frame of the ß-subunit of rLH (rLHß) was directly
amplified from the rat pituitary polyadenylated RNA by the RT-PCR
technique, using oligonucleotide primers
(5'-GATAAGCTTAAATGGAGAGGCTCCAGGG-3' and
5'-ATCTCGAGGAAGAGGAGAAGGCCGGGGA-3'). This cDNA fragment was then
subcloned between the HindIII and XhoI sites of
pcDNA3 vector (Invitrogen, San Diego, USA), and its sequence was
determined and confirmed to be identical with the reported sequences of
rLHß (24).
The partial cDNA sequences of CgA and SgII were also amplified from the rat pituitary polyadenylated RNA by the RT-PCR technique. Two sets of oligonucleotide primers (5'-CCAAGCTTAGTGCTCCCACTGGTGCAGAG-3' and 5'-ATCCTCGAGCTCCTGGCCCTTCCC-3' for a CgA fragment, and 5'-GATAAGCTTACAATATAAGACAGAGG-3' and 5'-ATCCTCGAGAACCCTCTCACGCTTCTGG-3' for a SgII fragment) were used for PCR, and a 1110-bp cDNA fragment of rat CgA (from position 40 of the nucleic acid sequence) (25) and a 693-bp cDNA fragment of rat SgII (from position 1 of the nucleic acid sequence) (26) were amplified. These cDNA fragments were subcloned into the HindIII and XhoI sites of pcDNA3 vector, and their sequences were determined and confirmed to be identical with the reported sequences of rat CgA or SgII.
Using these vectors inserted with LHß, CgA, and SgII cDNA fragments as templates, digoxigenin-labeled single strand RNA probes were prepared. Digoxigenin-UTP-labeled sense and antisense RNA probes were prepared using the DIG RNA Labeling Kit (Boehringer Mannheim Biochemica, Mannheim, Germany) according to the manufacturer’s recommendations.
Northern blot analysis
For Northern blot analysis, five rats in each experimental group
were killed by decapitation under anesthesia with pentobarbital (25
mg/kg, ip). Pituitaries and adrenals were immediately excised, quickly
frozen in liquid nitrogen, and stored at -70 C until used.
Total RNA of pituitary and adrenal glands from each rat (n = 5/experimental group) was individually extracted according to the procedure of Chomczynski and Sacchi (27). Ten micrograms of total RNA were separated on a 1.5% agarose formaldehyde denaturing gel (28). After the RNA was transferred to nylon membrane filters (GeneScreen Plus, DuPont-New England Nuclear Research Products, Boston, MA), the filters were baked for 2 h at 80 C and prehybridized for 4 h at 65 C in a hybridization solution containing 50% formamide, 5 x SSPE (0.75 M NaCl, 0.05 M NaH2PO4, and 5 mM EDTA, pH 7.4), 5 x Denhardt’s solution, 1% SDS, and 10% sodium dextran sulfate (mol wt, 500,000). Hybridization proceeded at 65 C for 16 h in the same buffer containing 100 ng/ml of each digoxigenin-11-UTP-labeled RNA probe. The filters were then washed in 2 x SSC (standard saline citrate)-0.1% SDS for 10 min at 25 C and in 0.2 x SSC-0.1% SDS twice at 65 C for 20 min. They were then washed briefly with TNE buffer (10 mM Tris-HCl, 500 mM NaCl, and 1 mM EDTA, pH 7.5) and treated with ribonuclease A (20 µg/ml) in the same buffer at 37 C for 20 min. After washing with TNE to remove ribonuclease, they were immersed in DIG buffer 1 (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 10 min.
Immunodetection of each hybridized digoxigenin-labeled RNA probe was performed using a DIG Luminescent Detection Kit (Boehringer Mannheim Biochemica) with some modifications. The filters were incubated with 1.0% blocking reagent in DIG buffer 1 for 60 min at 25 C and further treated with 0.075 U/ml polyclonal sheep antidigoxigenin Fab fragments conjugated to alkaline phosphatase in DIG buffer 1 for 30 min at 25 C. Excess antibody was removed by washing with DIG buffer 1 containing 0.2% Tween-20 for 15 min twice. The washed filters were twice equilibrated for 5 min with DIG buffer 3 (100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl2, pH 9.5) and assay buffer (100 mM diethanolamine and 2 mM MgCl2) for 3 min each, and then incubated in 0.1 mg/ml disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (Tropix, Bedford, MA) in assay buffer for 10 min at 25 C. After excess substrate was removed, the filters were exposed to x-ray film for 30 min and developed.
Tissue preparation for electron microscopy
For routine electron microscopy, three rats from each
experimental group were anesthetized with pentobarbital (25 mg/kg, ip),
perfused with 50 ml physiological saline and then with 250 ml 2%
glutaraldehyde-2% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.2. The pituitaries were quickly excised, cut into small
pieces, and immersed in the same fixative at 4 C for 24 h. After
washing thoroughly with 0.1 M phosphate buffer, pH 7.2,
containing 7.5% sucrose, samples were postfixed with 1%
OsO4 in 0.1 M phosphate buffer containing 7.5%
sucrose (pH 7.2) at 4 C for 2 h. After postfixation, the samples
were dehydrated using graded alcohols and embedded in Epon 812. For
immunocytochemistry, rats (n = 3/group) were perfused with
physiological saline and 4% p-formaldehyde in 0.1
M phosphate buffer containing 4% sucrose (pH 7.2). After
fixation by perfusion, the pituitaries were excised, cut into small
pieces, dehydrated in graded alcohols without postfixation by
OsO4, and embedded in Epon 812.
Immunocytochemistry
Ultrathin sections from tissue blocks embedded in Epon 812 were
cut with an ultramicrotome (Ultracut, Leica, Nussloch, Germany) and
mounted on nickel grids. The sections were etched with 1% sodium
methoxide for 30 sec before the following immunostaining procedures
(29). They were incubated with 5% nonimmune goat serum for blocking at
25 C for 20 min, and then further incubated with the following first
antibodies at 4 C for 12 h: anti-oLHß (diluted 1:100 for
osmicated samples and 1:500 for unosmicated samples), anti-rCgA
(diluted 1:200), anti-rSgII (diluted 1:200), or antirat cathepsin D (13
µg/ml). They were then treated with gold-labeled goat antirabbit IgG
for 1 h at 25 C (gold particles of 15 or 8 nm in diameter); the
size adjustment and labeling of colloidal gold particles were performed
according to the method of Slot and Geuze (30). Between each step, the
grids were washed in 0.02 M Tris-HCl-buffered 0.5
M saline, pH 8.2, containing 0.1% BSA. For double
immunostaining, the two-face technique of Bendayan (31) was applied
(sizes of gold particles, 8 and 15 nm). After the immunoreactions, the
sections were contrasted with saturated aqueous solutions of uranyl
acetate and lead citrate and examined with a Hitachi (Tokyo, Japan)
H-7100 electron microscope.
Morphometry
After double immunostaining, the cytoplasmic areas of pituitary
cells that were immunoreactive for LH and CgA or for LH and SgII were
randomly photographed at a magnification of x12,000 (n =
20/experimental group). From the samples of castrated rats with no
steroid replacement (group B), only the typical castration cells
containing extremely dilated rough endoplasmic reticulum (rER) were
analyzed in this way. The profile sizes of the secretory granules were
measured with NIH Image software (written by W. Rasband, NIH, Bethesda,
MD), and the results were statistically analyzed with KaleidaGraph
software (Synergy Software, Reading, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of CgA and SgII in the pituitary after castration and
sex steroid replacement
To determine whether the biosynthesis and storage of CgA and SgII
in pituitary glands are affected by estradiol and testosterone,
Northern blot and immunoblot analyses were performed in pituitary
samples.
As shown in Fig. 1
, the messenger
RNA (mRNA) level of LH ß-subunit in the pituitary gland was
drastically increased after castration, whereas the replacement of
testosterone or estradiol to the castrated rats markedly suppressed the
expression of LHß below the control level. Castration and
simultaneous replacement of testosterone had no effect on the
expression level of CgA in the pituitary, whereas replacement with
estradiol drastically suppressed its level. The mRNA level of SgII
clearly increased after castration and decreased slightly with sex
steroid replacement. Estradiol appeared to decrease SgII expression
more effectively than testosterone, but these suppressive effects were
not as drastic as those on CgA expression. The mRNA levels of CgA and
SgII in adrenals obtained from the same animals appeared to be constant
and were not significantly influenced by castration or accompanying sex
steroid replacement.
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). The amount of CgA in the pituitary of
the castrated rats was less than that in control rats. Interestingly,
testosterone and estradiol exhibited opposite effects on the storage of
CgA in the pituitary gland after castration; after testosterone
replacement, the amount of CgA in the pituitary was raised above the
level in castrated animals. On the contrary, CgA was almost completely
depleted from the pituitary after estradiol replacement. In addition,
the amount of SgII in the pituitary remained constant regardless of the
treatment received, except that it was slightly elevated after
castration alone. In adrenals, the amounts of CgA and SgII were also
examined by immunoblotting and were not affected by castration alone or
simultaneous sex steroid replacement (data not shown).
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Eight weeks after castration, hyperstimulated gonadotropes, called
castration cells or signet ring cells (32, 33), were prominent in the
anterior pituitary. They contained extremely dilated rough ER, well
developed Golgi complex, and numerous secretory granules (Fig. 3a). Most secretory granules in the
castration cells were small and moderately electron dense and were
intensively labeled by Immunogold particles, indicative of the presence
of LH. Immunoreactivity for CgA was faint or undetectable in these
secretory granules (Fig. 3b), whereas significant labeling for SgII was
detected (Fig. 3c). Sporadically, a few extra large granules were
observed among the small granules, but these appeared to be lysosomes,
as they showed immunoreactivity for lysosomal enzymes and not for CgA
or SgII (Fig. 3d). As previously reported (13, 17, 18), some other
types of gonadotropes could be distinguished among typical castration
cells in the anterior pituitary. Additional details of the
heterogeneity in male rat gonadotropes, especially on the
immunocytochemical localization of CgA and SgII within them, have been
described previously (14).
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). Immunoreactivity for CgA was well
detected in large granules (Fig. 4b), whereas that for SgII was
restricted in the small granules (Fig. 4c). Double immunostaining
clearly demonstrated that CgA and SgII were exclusively segregated in
large and small granules, respectively, as observed in the typical
gonadotropes of untreated control male rats (Fig. 5).
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), which were
colabeled with Immunogold particles, indicative of the presence of SgII
(Fig. 6c). Immunogold particles showing CgA, which are usually
associated with large secretory granules, were no longer detected in
the cytoplasm of the cells (Fig. 6b). Extra large granules were
occasionally observed among the small granules. These extra large
granules also appeared to be lysosomes, as they were immunostained by
anticathepsin D (data not shown).
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).
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On the other hand, secretory granules that contained SgII were regularly observed in gonadotropes, but the mean profile diameters of these varied depending on the treatment received; the diameter of the secretory granules that contained SgII in the castration cells was the largest (156.4 ± 0.7 nm), whereas that in estradiol-treated rats was the smallest (111.8 ± 0.5 nm). The mean diameters of SgII-positive secretory granules in gonadotropes of nontreated control and testosterone-treated rats were similar to one another (130.8 ± 0.5 and 135.1 ± 0.6 nm, respectively) and intermediate in size between those in castration cells and in the gonadotropes of estradiol-treated rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As demonstrated in our previous studies, CgA and SgII are separately localized to large and small secretory granules of gonadotropes (12, 13, 14). The present study clearly shows that the appearance of large secretory granules in gonadotropes is closely related to the synthesis and storage levels of CgA in the anterior pituitary, which appears to be differently regulated by androgen and estrogen. We now consider two main aspects of our findings: 1) the regulation of granin synthesis by steroids, and 2) the significance of granins for the biogenesis of secretory granules in gonadotropes.
Regulation of granin synthesis by sex steroids
In addition to hormones, such as LHRH and gonadotropins, the
expression of granins in the pituitary gland has been shown to be
affected by steroid hormones. Among the various steroid hormones, the
effects of estradiol on the expression of granins in the anterior
pituitary have been extensively analyzed by both in vivo
(34, 35, 36, 37) and in vitro (38, 39) studies. The in
vivo studies have generally indicated that the expression of CgA
in the pituitary gland is increased after ovariectomy and drastically
suppressed by simultaneous estradiol replacement (34, 35, 36). These
findings have also been confirmed by an in vitro study using
cultured pituitary cell aggregates, suggesting that estradiol directly
suppresses CgA expression in pituitary cells (39). In the present
study, castration alone had no effect on the mRNA level of CgA in the
male rat pituitary, but the suppressive effects of estradiol on the
expression and storage levels of CgA were observed in the pituitary as
other researchers have reported (34, 35, 36). Moreover, our present
morphological and immunocytochemical findings clearly demonstrate that
the large secretory granules containing CgA appeared only in
gonadotropes and vanished from these cells in parallel with the
depletion of CgA in the pituitary by estradiol replacement. These
findings suggest that estradiol suppresses the expression level of CgA,
specifically in gonadotropes among the various pituitary endocrine
cells.
We also found that estradiol showed inhibitory effects on the expression of SgII, but to a lesser extent than on that of CgA. This result is consistent with in vivo data in which the increased level of SgII mRNA after ovariectomy has been shown to be decreased to the level in control female rats by accompanying estradiol replacement (34, 36, 37). Moreover, Anouar and Duval, using primary cultures of pituitary cell aggregates, demonstrated that estradiol has inhibitory effects on the expression of SgII (39). A report also exists that concludes that estradiol could slightly up-regulate SgII expression in the pituitary under certain in vivo conditions (35). In any case, however, the effect of estradiol on the expression of SgII in the pituitary is likely to be much less drastic than that on the expression of CgA.
In contrast to investigations of the effects of estradiol, no appropriate study has yet appeared concerning the effect of testosterone on the regulation of granin expression in the pituitary. As shown in the present study, the mRNA level of CgA was not affected by castration and simultaneous testosterone replacement. The expression level of SgII was increased after castration, but was restored to the normal level of untreated control male rats by testosterone replacement. Compared with the effects of estradiol, testosterone showed no effect on CgA expression and had a weak suppressive effect on SgII expression, although the dose of testosterone used was sufficiently high to suppress LH ß-subunit expression. An in vitro study using primary culture of gonadotropes will be necessary to assess the direct effect of testosterone on granin expression.
In addition to gonadal sex steroids, dexamethasone, a synthetic glucocorticoid, is known to increase CgA expression in the pituitary gland of male rats (40) and in cultured pituitary cell aggregates (39). In addition, adrenalectomy markedly decreases the expression of CgA in pituitary glands (41). From the evidence now available, it seems likely that glucocorticoid maintains or up-regulates the expression level of CgA in pituitary glands. This may be explained by analyses of the promoter region of the CgA gene (42, 43).
The present findings demonstrate that large secretory granules containing CgA were well preserved in gonadotropes of both the control and testosterone-treated castrated male rats, but not in those of estradiol-treated rats. Under the influence of estrogen, the expression of CgA in gonadotropes was suppressed, resulting in the disappearance of secretory granules containing CgA from gonadotropes. On the contrary, when the influence of estrogen is negligible, the expression of CgA in gonadotropes might be maintained by glucocorticoid or androgen. The results in the present study are entirely consistent with a previous report that concluded that the expression level of CgA in the female rat pituitary is significantly lower than that in the male rat pituitary (35).
In addition to the effects of sex steroids on the regulation of granin synthesis, androgen might also contribute to a negative feedback effect on LHRH release from the hypothalamus, resulting in the retention of secretory granules containing CgA in gonadotropes. Without this effect, large secretory granules could not be stored in gonadotropes despite the high level of CgA expression, as seen in the castration cells. In any case, our experimental findings provide an appropriate explanation for the sex-related differences in the ultrastructure of secretory granules within gonadotropes, as previously reported (16, 17, 18).
Granins and the biogenesis of secretory granules in
gonadotropes
Although several extracellular or intracellular functions have
been proposed for the granins in neuroendocrine cells, the
interpretation of their physiological roles remain controversial (for
reviews, see Refs. 1–4). Among the various putative intracellular
roles, it has been suggested that the granins may contribute to the
aggregation and packaging process of regulated secretory proteins in
the trans-Golgi network.
The present study shows that large secretory granules in gonadotropes disappeared when the expression of CgA was completely suppressed by estradiol replacement after castration. In contrast, testosterone replacement in castrated rats resulted in the presence of gonadotropes that exhibited large and small secretory granules comparable to those in untreated controls, although the immunoreactivity for LHß was largely reduced by the negative feedback of testosterone. To our knowledge, the present study is the first report that clearly demonstrates a close association between the presence of a particular type of secretory granule in endocrine cells and the expression level of a corresponding granin(s).
It has been shown that purified granins spontaneously aggregate in a relatively low pH range (pH 5–6) in the presence of appropriate concentrations of calcium ions (10–20 mM) (44, 45). This has been confirmed by studies using a cell-free system (46, 47, 48). A more recent in vitro study has demonstrated that LH coaggregates with granins but does not show self-aggregation, even under low pH and high calcium conditions (49). Although certain other hormones, such as PRL, self-aggregate without participation of granins (49, 50), it seems likely that CgA and SgII actively participate at least in the packaging process of gonadotropins into secretory granules.
In summary, the present study shows that the expression levels of CgA and SgII in the pituitary are affected differently by male and female sex steroids, and that the appearance of large secretory granules in pituitary gonadotropes corresponds well with the expression and storage levels of CgA. These different effects of androgen and estrogen on the expression levels of CgA may contribute to the sex-related differences in the ultrastructure of secretory granules within gonadotropes. In addition, we were able to confirm that CgA and SgII are separately sorted to large and small secretory granules of gonadotropes in the male rat anterior pituitary even after castration and accompanying replacement of testosterone. Further studies are required to explain how CgA and SgII are selectively sorted into the two secretory granules in gonadotropes, and why these cells require two subsets of secretory granules.
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Acknowledgments |
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Footnotes |
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Received January 23, 1998.
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