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Division of Endocrinology and Metabolism (S.K., S.J.W.), Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; and Division of Endocrinology and Metabolism (Y.F., Y.O., S.J.W.), University of Louisville, Louisville, Kentucky 40202
Address all correspondence and requests for reprints to: Dr. Stephen J. Winters, Division of Endocrinology and Metabolism, Department of Medicine, University of Louisville, ACB-A3G11, 550 Jackson Street, Louisville, Kentucky 40202. E-mail: . sjwint01{at}gwise.louisville.edu
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Abstract |
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Introduction |
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Most of what is known about the cellular and molecular mechanisms for the paracrine regulation of FSH biosynthesis and secretion is from experiments conducted with rats and rat pituitary cells and, more recently, from transgenic mouse models. The applicability of all findings to primates (including humans) is not certain, however, because there are well-established, fundamental differences in gonadotropin secretion between rodents and higher primates. For example, there is a dramatic increase in FSH secretion after bilateral orchidectomy in men (9) and in the nonhuman male primate (10) that correlates in the primate model with a 40-fold rise in FSH-ß gene expression (10). By contrast, there is, at most, a 2- to 3-fold rise in FSH-ß mRNA levels and FSH secretion after castration in adult male rats (11). Moreover, there a postcastration rise in pituitary follistatin expression in rats (12) but not in monkeys (13), and the time course of those changes in rats suggests that follistatin acts to restrain FSH-ß gene expression after orchidectomy in that species. In addition, T stimulates FSH-ß mRNA and FSH secretion in rats in the absence of GnRH (14, 15) and in cultured rat pituitary cells (16, 17) in which T suppresses follistatin gene expression (6). Thus, follistatin suppression correlates with, and may mediate, the T-induced rise in FSH-ß mRNA levels in rats. On the other hand, the combination of T and a GnRH antagonist reduced more effectively serum FSH levels in men than did the GnRH antagonist alone (18), and T enanthate suppressed circulating FSH levels in untreated GnRH-deficient men (19). Androgens also decrease human FSH-ß mRNA levels in pituitary cells from mice expressing this human transgene (20), implying that species-specific factors in the pituitary cellular environment, rather than motifs in the FSH-ß promoter, explain differences in FSH regulation by T in rodents compared with primates.
To gain further insight into the cellular and molecular mechanisms governing FSH-ß gene expression in primates, we have established primary pituitary cell cultures from adult male rhesus monkeys. Using this model, T and dihydro-T, which effectively suppress GnRH-stimulated LH secretion and decrease 
-subunit mRNA levels in pituitary cells from adult male rats, were ineffective in pituitary cells from adult male monkeys (21). The present study explores the hypothesis that differences in the regulation of follistatin gene expression explain the dissimilarities in pituitary FSH-ß gene expression and FSH secretion after orchidectomy and T treatment in male rats and primates. Because the mRNAs that encode the FSH-ß subunit and follistatin are expressed at low levels in these cultures, pursuit of this hypothesis required the development of sensitive, quantitative assays for these mRNAs. We therefore developed quantitative competitive RT-PCR assays for FSH-ß subunit and follistatin mRNAs, and we applied these assays to RNA from primate pituitary cells in monolayer culture.
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Materials and Methods |
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3 Prime Inc. (Boulder, CO); MetaPhor agarose from FMC BioProducts (Rockland, ME); Titan One Tube RT-PCR System from Roche Molecular Biochemicals (Indianapolis, IN); and Lab-Tek chamber slides from Nalge Nunc International (Naperville, IL).
Pituitary cell cultures
Pituitary glands (generally two per experiment) from adult male rhesus monkeys (Macaca mulatta) were obtained from Covance Laboratories, Inc. Research Primates (Alice, TX) and were transported, on ice, to the laboratory. Anterior pituitary cells were dispersed enzymatically as previously described (21) and were allowed to attach onto 6-well culture plates at a density of 0.6–0.8 x 106 cells/well in 3 ml DMEM containing 10% FCS (serum-containing media). Twenty-four hours later, culture media were removed, and cells were washed once with, and incubated in, fresh serum-containing or serum-free media. The day of cell plating was designated as d 0. The cell yield was 2.1 ± 0.4 x 106 cells/pituitary, and cell viability was 96 ± 1% based upon trypan blue dye exclusion. All cultures were maintained at 37 C in a humidified atmosphere of 5% CO2-95% air.
Pituitary-derived fibroblasts
When monkey pituitary cells were dispersed, fibrous tissue debris remained, even after 60 min of enzymatic digestion. This fibrous tissue was used to establish pituitary-derived fibroblast strains as described previously (22). Fragments of tissue were placed on 10-cm tissue culture dishes (4–6/dish), and cultured at 37 C in DMEM containing 10% FCS. After 5–7 d, growth of spindle-shaped cells from the tissue fragments was first apparent, and cultures became confluent after 3–4 wk. Confluent cultures were removed from the dishes by exposure to Dulbecco’s PBS containing 0.25% trypsin and 0.02% EDTA, rinsed once with serum-containing media, and were subcultured in 75-cm2 culture flasks at a 1:4 split ratio. Pituitary fibroblasts were maintained in 75-cm2 culture flasks, in serum-containing media that was changed every 3–4 d. Cells at passage 3–5 were used for experiments.
Immunocytochemistry
The morphological appearance of the primary cultures was observed daily under a phase-contrast microscope. For immunocytochemical characterization, cells at a density of 0.05 x 106 cells/well were plated on Lab-Tek chamber slides coated with poly-L-lysine in 500 µl serum-containing media. Pituitary cultures were maintained for up to 5 d, and pituitary fibroblasts were cultured for 2–3 d. After washing with 50 mM PBS, cultures were fixed with 4% paraformaldehyde in 50 mM PBS for 30 min–2 h at room temperature. Immunostaining was performed using a rabbit polyclonal antiserum against bovine S-100 protein at 1:1,000 dilution (DAKO Corp., Carpentia, CA), a mouse monoclonal antihuman fibroblast surface protein antibody (clone number 1B10, Sigma) at 1:1,000 dilution, rabbit anticynomolgus monkey LH (NIH-AFP342994) at 1:20,000 dilution, and monoclonal antihuman CD83 at 1:10 to 1:50 dilution (BD; PharMingen, San Diego, CA). To reduce nonspecific binding, primary and secondary antisera were diluted in 50 mM PBS containing 0.05% Triton-X and 1% normal serum from the appropriate animal. Slides were incubated with primary antisera for 36–48 h at 4 C, washed thoroughly with 50 mM PBS, incubated for 1 h with the appropriate biotinylated secondary antisera (Vector Laboratories, Inc., Burlingame, CA; 1:1,000 dilution), washed thoroughly with 50 mM PBS, and processed with Vectastain Elite ABC Kit (Vector Laboratories, Inc.).
RNA sample preparation
Total RNA was extracted from the cell cultures using the guanidinium thiocyanate-phenol-chloroform procedure (23).
Competitive RT-PCR for FSH-ß subunit gene
A quantitative RT-PCR assay for FSH-ß subunit mRNA, using a synthesized competitive template (CT) RNA, was developed to measure the low levels of FSH-ß mRNA in primary cultures from male monkeys. The cynomolgus monkey FSH-ß cDNA (approximately 2000 bp) was generously provided by Dr. Scott Chappel and Christie Kelton of Ares Advanced Technology (Randolph, MA) and was cloned into the PstI site of pBR322 plasmid by the G/C tailing method. A CT standard that differs slightly in molecular weight from the native sequence was developed by adding a nonhomologous spacer fragment from pBR322 (Fig. 1A
). A 162-bp fragment [nucleotides (nt) 145–307, corresponding to a part of exon 1 and exon 2] was excised from the FSH-ß cDNA by DraIII digestion; and a 383-bp DNA fragment, derived from a MvaI digest of pBR322, was inserted to produce a larger PCR product that could be distinguished from the native product after agarose gel electrophoresis. After excision by PstI digestion, the recombinant cDNA fragment (1071 bp) containing the wild-type primer sequences and the nonspecific spacer sequence was subcloned into the PstI site of pSP64 poly(A) vector. The plasmid was linearized by EcoRI, treated with proteinase K, phenol-extracted, and used as a template in sense RNA (CT RNA) synthesis using the MAXIscript in vitro transcription kit with SP6 RNA polymerase. Synthesized CT RNA was treated with deoxyribonuclease I to remove template DNA; separated on a formamide agarose gel; eluted from the agarose; extracted with phenol once, phenol-chlorform-isoamyl alcohol once, and chlorform-isoamyl alcohol once; and precipitated with 100% ethanol. The RNA pellet was washed twice with 75% ethanol, dried briefly, and reconstituted with diethyl pyrocarbonate-treated water. After determination of concentration and purity, the CT RNA was spiked with 1 U/50 ml RNase inhibitor, and aliquots were stored in siliconized microcentrifuge tubes at -70 C.
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A constant amount of sample RNA (200 ng) was combined with decreasing amounts of the CT RNA (800, 80, 8, and 0.8 pg) in a 40-µl reaction volume. The reaction, using the Titan One Tube RT-PCR System (Roche Molecular Biochemicals), was optimized in preliminary studies for the following reagents: 1.5 mm MgCl2, 5 mm dithiothreitol, 0.4 mm of primers SK96–298 and SK96–299, 200 mm deoxynucleotide triphosphates, and 1 U RNase inhibitor. Samples were amplified under the following temperature profile: RT at 50 C for 30 min; initial denaturation at 94 C for 3 min; followed by 28 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and extension at 68 C for 45 sec; with a final extension at 68 C for 7 min. Bands on ethidium bromide-stained gels (Fig. 1B
) were corrected for differences in base pair length between the native and CT RNA-derived native and CT PCR products, which is 262/483 = 0.542. The percentage of native product in each RT-PCR reaction was determined using the equation: % Native = [ODnative/(ODCTx0.542 + ODnative)]x100.
As shown in Fig. 1C, a regression line with the equation y = AxLog10(x) + B was generated for each set of four RT-PCR reactions by plotting the percent native product in each reaction vs. the Log10{CT (pg/reaction)}. Solving this equation for y = 0.5 (half the DNA was the native product) allows for a calculation of the amount of native mRNA in the sample. Preliminary assays were conducted to optimize the amount of sample RNA and the number of PCR cycles so that products were within the exponential phase of amplification. PCR primers were designed to span an intron to control for amplification of genomic DNA carry-over, and no detectable amplification from genomic DNA was observed on the gels. To control for interassay variability, aliquots of a pool of total RNA samples were assayed. To verify the absence of contaminants, periodic control experiments omitting RNA were also conducted. To validate the accuracy of quantification, FSH-ß mRNA levels in pituitary RNA samples from five orchidectomized adult monkeys were determined by two methods: RT-PCR and Northern analysis. The correlation coefficient relating the results for these two methods of analysis was 0.92 and 0.96, respectively, in replicate assays.
Quantitative competitive RT-PCR for follistatin mRNA
Total follistatin mRNA levels (a sum of the two alternative splicing variants of follistatin mRNA, FS-288 and FS-315) were determined as previously described (22).
Immunoassays
FSH levels in culture media were estimated using homologous RIA reagents (26). Recombinant cynomolgus FSH (NICHHD Rec-MoFSH-RP-1, AFP-6940A) was the reference preparation and the radioiodinated tracer, and a polyclonal rabbit antiserum (AFP782594) against recombinant cynomolgus FSH was used as the primary antibody. The 50% intercept of the assay was 0.06 ng AFP-6940A/tube. Cross-reactivity with recombinant cynomolgus LH (AFP-6936A) and the common -subunit (AFP-5679A-SIAFP) was less than 0.02% and less than 0.01%, respectively. The minimal detectable dose was 6 pg/tube. The intra- and interassay coefficients of variation were less than 5% and 8%, respectively. The concentration of monkey LH in culture media was determined as previously reported (21).
The concentration of follistatin in culture media was determined using ELISA reagents that were generously provided by Dr. Nigel Groome, as described previously (22). Media were concentrated 10-fold using a Speed-Vac concentrator (Savant Instruments, Farmingdale, NY) before assay.
Experimental design
Exp 1. A comparison of the effects of serum and growth factors on pituitary cell- and fibroblast-cultures.
The microscopic appearance, immunocytochemical characteristics, and total RNA recovered on d 1–4 in culture were compared in monkey pituitary cells cultured in serum-containing medium or in one of the two following serum-free, chemically defined media beginning on d 1: serum-free DMEM (DMEM supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 50 nm sodium selenite, 2.5 ng/ml epidermal growth factor, 0.5 nm triiodothyronine, and 20 µm putrescine); or serum-free D-valine-replaced DMEM/F-12 (D-valine replaced DMEM/F-12 supplemented with the same additives as the serum-free DMEM). The growth effects of human granulocyte-macrophage-colony-stimulating factor (GM-CSF), at doses of 250 or 1,000 U/ml, on cells cultured in serum-containing or in serum-free D-valine-replaced DMEM were also examined.
Pituitary fibroblasts at the third passage were plated onto 6-cm culture dishes at 0.3 x 106 cells/dish in 3 ml serum-containing medium. Cultures were washed with, and incubated in, fresh DMEM containing 10% FCS (control) or 0.25% BSA (serum-depleted) medium, beginning 48 h after plating. At 120 h after plating, both control and serum-depleted cultures were washed with, and further incubated in, fresh DMEM containing 10% FCS. Cells were harvested at 48, 72, 96, 120, 128, 144, and 168 h after plating by treatment with trypsin, and the number of cells was counted using a hemacytometer.
Exp 2. Regulation of FSH and follistatin in monkey pituitary cultures.
Time-dependent changes in FSH-ß and follistatin mRNA levels and in FSH secretion were determined in experiments (n = 5), in which cultured pituitary cells were grown in serum-free D-valine-replaced DMEM/F-12 from d 1–3. Twenty-four hours after plating, culture media were removed, and cultures were washed twice with control medium. Then, cultures were treated with the following test agents: PACAP (10 nm), GnRHa (10 nm), rh activin-A (3 ng/ml), or T (20 nM), or with control medium for 8 h or 48 h. Media were saved for immunoassay, and cells were harvested for extraction of total RNA.
Data analysis
Data are presented as the mean ± SEM. Because of cell growth characteristics, mRNA levels and the amount of hormones secreted varied in vehicle-treated cultures in replicate experiments. Therefore, in each replicate experiment, the results for hormone-treated cells were expressed as a percentage of the corresponding value for vehicle-treated cells. Results were then compiled for statistical analysis. Multiple group differences were analyzed by ANOVA and post hoc Dunnet’s test; t test for unpaired data was used to compare results between two groups.
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). Thereafter, flattened, polygonal cells with long cytoplasmic processes began to proliferate as early as d 2. Consequently, the primary cultures on d 3 consisted of independent round cells as well as small colonies of flattened cells. There was further proliferation and clustering of flattened cells by d 5, although round, refractile cells could still be distinguished.
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indicates that, in cultures grown either in serum or in serum-free media, the total amount of RNA recovered increased with time in culture. In addition, the growth characteristics and microscopic appearance of the rapidly proliferating polygonal cells in the primary cultures in serum-free media containing putrescine, and D-valine rather than L-valine, were essentially the same as in serum-containing media (not shown).
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Immunocytochemical studies
In Fig. 3, anterior pituitary cell cultures were examined by immunocytochemical staining for LH, for S-100 protein, a marker of folliculostellate cells (27), and for antifibroblast surface antigen 1B10, which reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes (28). In cultures on d 1, approximately 10% of the round, refractile cells stained positive for LH, with no increase on d 3. The proliferating cells, which were evident by d 3 in culture, were specifically and intensely stained with S-100 antisera in both the cytoplasm and nucleus. Several S-100-positive cells were also noted in the d-1 cultures, although those cells were indistinguishable in shape, at that time, from the refractile LH-positive cells. There were no 1B10-positive cells through d 5. Omission of each of the primary antisera resulted in minimal background staining. In addition, preabsorption of the S-100 antisera with 1 or 10 µg purified bovine S-100 protein (Calbiochem, La Jolla, CA), for 24 h before its application to the slides, eliminated immunolabeling (not shown).
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). These cells reacted positively to the 1B10 antibody predominantly throughout the cytoplasm, leaving the nuclei as punched-out negative regions. This staining pattern for the 1B10 antibody is similar to that of cultured human skin fibroblasts (22). Omission of the primary antiserum resulted in negligible background staining (not shown). With the S-100 antiserum, only minimal background staining was observed.
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Because folliculostellate cells from other species produce cytokines and growth factors, and because of their fluid shape, they may be related to dendritic cells of the immune system (29). Accordingly, monkey pituitary cells in d-3 cultures were stained for CD83, a marker for primate dendritic cells (30). All pituitary cells were CD83-negative using 1:10 dilution of A/S (not shown). Moreover, adding GM-CSF to the cultures, which accelerates growth of dendritic cells (31), failed to stimulate proliferation of S-100-positive cells (not shown).
Follistatin and FSH-ß gene expression and FSH secretion by primary pituitary cell cultures
To begin to study the hormonal interactions between primate pituitary folliculostellate cells and gonadotrophs, FSH secretion and FSH-ß subunit and follistatin mRNA levels were determined in pituitary cells cultured in serum-free D-valine-replaced media for up to 3 d. This medium was selected to limit fibroblast growth by the cultures because fibroblasts also express follistatin (22). As shown in Fig. 5, follistatin mRNA levels increased, from d 1–3 in culture, by 380 ± 78% (P < 0.01). This increase markedly exceeded the rise in total RNA that was 57 ± 10% (P < 0.001). By contrast, FSH-ß subunit mRNA levels on d 3 declined to 49 ± 16% of the value for d 1 cultures (P < 0.05). FSH secretion also decreased to 71 ± 10% (P < 0.05) from d 1–3 in culture.
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summarize experiments in which cells on d 1 or d 3 in culture were stimulated for 8 h with control media or with media containing 3 ng/ml rh-activin-A, 10 nM GnRHa, 10 nM PACAP38, or 20 nM T. Follistatin mRNA levels were increased by activin, PACAP, and T, but not by GnRHa. Moreover, GnRHa, activin, and PACAP increased the level of FSH-ß mRNA in monkey pituitary cultures. On the other hand, FSH-ß levels were suppressed by T.
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). Moreover, FSH secretion was stimulated by activin-A and by GnRHa but was unaffected by PACAP or T.
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Discussion |
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In 1974, Tang and Spies (32) noted that the spherical endocrine cells in monolayer cultures from the anterior pituitary of female cynomolgus monkeys were overgrown by flattened epithelioid cells, within 6 d, when media were supplemented with monkey serum. Subsequently, Bethea (33) reported that undifferentiated fibroblast-like cells overgrew the endocrine cells in monkey anterior pituitary cell cultures within 8 d, using serum-containing media; whereas, in serum-free media, these cells had a rounder, more compact shape. Neither report characterized those dividing cells further, however. Proliferating cells have also been noted in primary rat (34) and ovine (35) pituitary cell cultures. Koch and Lutz-Bucher (36) concluded that the proliferating cells in rat pituitary cultures were pituitary fibroblasts because of their morphology and vimentin-positive immunoreactivity, but those authors noted that folliculostellate cells are also vimentin positive (37). Pituitary folliculostellate cell proliferation also occurs in rats in vivo when pituitary tissue is grafted beneath the renal capsule (38).
Folliculostellate cells were first identified by electron microscopy by Rinehart and Farquhar (39) in 1953, and highly enriched folliculostellate cell cultures were reported in 1986 by Ferrara et al. (40) from bovine pituitaries. Folliculostellate cells can be identified by immunostaining for S-100 protein (41), and they account for 5–10% of anterior pituitary cells in the rat pituitary (39). These stellate-shaped cells encircle well-defined cavities and extend cytoplasmic processes (42) that connect with other folliculostellate cells by gap-junction channels (43). Folliculostellate cells were recently shown to communicate by calcium waves (44). But neither the origin nor the function of folliculostellate cells is well understood. There is evidence that some folliculostellate cells are related to dendritic cells of the immune response (29), whereas neuroectodermal origin is suggested because neuroglial cells also express S-100 protein (45). Like pituitary endocrine secretory cells, folliculostellate cells express Ptx-1 (46), suggesting that they could also be derived from a pituitary stem cell. The folliculostellate cells identified in the present primate pituitary cell cultures were immunonegative for CD83, a marker for primate dendritic cells (30), and cell proliferation was unaffected by GM-CSF. Thus, monkey folliculostellate cells do not behave like mature dendritic cells in culture, and their lineages may be unrelated.
There is accumulating evidence that folliculostellate cells serve a paracrine function within the pituitary. Folliculostellate cells influence the function of lactotrophs (47) and gonadotrophs (48) and perform immune functions (29). Products of folliculostellate cells that are likely to influence surrounding endocrine cells include basic fibroblast growth factor (49), vascular epithelial growth factor (50) and leukemia inhibitory factor in the cow (51), interleukin-6 in the mouse (52), nitric oxide synthetase (53) and lipocortin-1 (54), and follistatin in the rat (48, 55) and cow (50). Which of these factors, in addition to follistatin, is expressed in primate folliculostellate cells remains to be established.
There was no effect of GnRHa on follistatin mRNA levels in cultured primate pituitary cells. Similarly, follistatin mRNA levels were unaffected by orchidectomy in adult male rhesus monkeys (13) in which GnRH expression is increased (56). Together, these findings imply that follistatin expression in the primate pituitary is not GnRH dependent. In rats, on the other hand, although all pituitary cell types may express the follistatin gene (57), follistatin mRNA levels rise after orchidectomy (12), and this increase can be blocked by a GnRH antagonist (4). In addition, the concentration of follistatin mRNA in cultured rat pituitary cells is increased by GnRH (6). Together, these findings implicate gonadotrophs as a major source of follistatin in rats. In other studies, follistatin protein in the human pituitary was limited to somatotrophs (58). Therefore, the pituitary cells that express follistatin may differ in primates and rats, and the parallel increase in folliculostellate cells and follistatin mRNA concentrations in monkey pituitary cell cultures suggests that these cells are a major source of follistatin in primates.
The reciprocal changes in follistatin and FSH-ß mRNA expression with time in culture are consistent with the known effects of follistatin to bind to and neutralize activin and to thereby decrease FSH-ß mRNA levels (59, 60), although it is possible that loss of gonadotrope cells also contributed to the fall in FSH-ß mRNA. This pronounced decline in FSH-ß-gene expression with time in culture contrasts strikingly with results in pituitary cultures from rats which continue to secrete FSH for several weeks (61), perhaps because of up-regulation of FSH-ß mRNA by activin (62). Thus, it follows that increased production of follistatin by monkey pituitary cells cultures would block this activin effect.
On the other hand, follistatin mRNA concentrations were increased by PACAP and activin in primate, as in rat pituitary cultures (6, 7). Folliculostelate cells are known to be targets for PACAP (63, 64). Using dual immunocytochemistry and in situ hybridization, we showed recently that follistatin gene expression was stimulated by PACAP in rat folliculostellate cells and in gonadotrophs (48). Danila et al. (65) described a folliculostellate cell line derived from a human pituitary tumor that expressed follistatin and was responsive to activin stimulation.
T increased follistatin mRNA levels and protein secretion by pituitary cultures from primates, whereas T reduced follistatin expression in rat pituitary cell cultures (6). Although the reason for this species difference in regulation is not known, it could relate to different cell types expressing follistatin. In addition, the increase in follistatin in T-stimulated pituitary cultures from primates was associated with a decline in FSH-ß mRNA levels, whereas T increases FSH-ß mRNA levels in rat pituitary cell cultures (11, 17), as well as in rats in vivo in the absence of GnRH (14, 15). Together, these data suggest that androgens regulate FSH-ß mRNA, in part, through a follistatin-related mechanism. Although FSH secretion by nonhuman primate pituitary cell cultures was not reduced by T, these are provocative findings because T production and serum follistatin levels (66) are increased in women with the polycystic ovary syndrome among whom there is a deficit in FSH production when compared with increased production of LH (67). Therefore, it is intriguing to speculate that increased pituitary follistatin expression could play a role in the gonadotropin disturbance in women with this disorder.
In summary, primary pituitary cell cultures from the nonhuman primate grown in serum-free or serum-containing DMEM are rapidly overgrown by epithelioid cells with the morphological, immunocytochemical, and proliferative characteristics of folliculostellate cells. Follistatin production increases with time in culture, and follistatin expression in these cultures is stimulated by activin, PACAP, and T, but not by GnRH. These data suggest that folliculostellate cells may be a major source of follistatin in primates and may play a role in the previously observed differences in the hormonal control of FSH-ß gene expression between rodents and primates.
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Acknowledgments |
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Footnotes |
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Abbreviations: CT, Competitive template; GM-CSF, granulocyte-macrophage-colony-stimulating factor; GnRHa, [D-Trp6]-GnRH ethylamide; nt, nucleotide; PACAP, pituitary adenylate cyclase-activating polypeptide; RNase, ribonuclease.
Received November 2, 2001.
Accepted for publication February 19, 2002.
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in a subpopulation of folliculostellate cells and in an unidentifiable population of non-hormone-secreting cells. J Histochem Cytochem 45:847–857This article has been cited by other articles:
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