| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article, PCC |
a Anatomisches Institut, Technische Universität München, D-80802 München, Germany
b I. Frauenklinik der Ludwig-Maximilians Universität München, D-80337 München, Germany
c Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot, Israel
ABSTRACT
The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.
cAMP, FSH, gene regulation, granulosa cells, ovary
INTRODUCTION
Ovarian development and function are subjected to regulation not only by pituitary hormones but also by intragonadal regulatory factors and neurotransmitters. In the monkey ovary, evidence was provided recently indicating that in addition to ovarian innervation, neuron-like cells are present. These cells express the genes for dopamine-ß-hydroxylase and tyrosine hydroxylase and thus may represent an intraovarian source of catecholamines [1, 2]. Currently, investigations are being performed to determine whether similar cells occur in ovaries of other species including rodents and humans. To this end, we sought to identify neuronal elements by their expression of neuron-specific proteins. One key element of the molecular machinery required for regulated exocytosis of neurotransmitters is the synaptosome-associated protein of 25 kDa (SNAP-25), which was initially described as a component of synapses [3]. According to the SNARE hypothesis (reviewed in [4]), SNAP-25 interacts with syntaxin and vesicular synaptobrevin to build the 7S core complex, which docks synaptic vesicles at the plasmalemma. This complex recruits two soluble proteins known to be involved in many membrane trafficking processes, N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (
-SNAP) forming a 20S complex. In the presence of ATP, the 20 S protein complex is disassembled, and exocytosis can be triggered by a calcium signal.
SNAP-25 and its partners participate also in regulated exocytosis of hormones by endocrine cells derived from the neuroectoderm. Examples are hormone secretion by the anterior pituitary [5], catecholamine release by chromaffin cells of the adrenal medulla [6], histamine secretion by enterochromaffin-like cells of the stomach [7] and insulin secretion by beta cells of the pancreatic islet [8].
The results presented in this study unexpectedly demonstrate that SNAP-25 is present in steroid-producing cells of the ovary, which are of mesenchymal origin, and in oocytes. Moreover, SNAP-25 expression in GC is regulated by FSH but not by LH. Thus, this key protein of exocytosis may play a major role in the function of steroidogenic cells of the ovary.
MATERIALS AND METHODS
Granulosa Cell Cultures and Tissue Samples
Follicular fluid-containing granulosa cells was derived from in vitro fertilization (IVF) patients. The experimental procedure and the use of the cells was approved by the local ethics committee, and the women gave their written consent. Isolation and culture of the cells were performed as described elsewhere [9], with only slight modifications. These included a Percoll centrifugation step (50%; Pharmacia, Freiburg, Germany), according to Pellicer et al. [10], that was performed to avoid red blood cell contamination. Cells were washed with Dulbecco modified Eagle medium (DMEM):Ham F12 medium (1:1) and seeded in the same medium with 10% fetal calf serum onto 35 or 60 mm Falcon (Nunc, Wiesbaden, Germany) culture dishes, and for immunocytochemistry, on Labtek cell culture chambers (Nunc). All culture dishes were coated with laminin (Sigma, Deissenhofen, Germany) as described elsewhere [11]. Cultures were kept in an incubator in a humidified atmosphere with 5% CO2 at 37°C. After 24 h, media were replaced, and nonadherent cells (mainly representing red blood cells) were removed by gentle washing. At this time, cells were used for experiments as described below or in some cases were kept longer, with daily medium changes.
The rat cell line GFSHR-17 was maintained in DMEM:Ham F12 (1:1, Sigma) containing 5% fetal calf serum [12]. All chemicals used in stimulation experiments were from Sigma except H89, which was obtained from Biomol (Hamburg, Germany). Cultures grown for at least 1 day were incubated with medium containing hCG (10 IU/ml), FSH (0.5 IU/ml), dibutyryl-cAMP (db-cAMP, 1 mM), or H89 (30 µM) and harvested for investigation after 24 h.
Paraffin-embedded normal ovarian tissue samples (n = 3) were from a tissue archive at the Women's Hospital in Munich. These samples had been collected from premenopausal women during autopsies. Adult cycling Sprague-Dawley rats were purchased from Charles River (Sulzfeld, Germany), housed under standard conditions, and decapitated under deep CO2 anesthesia as described elsewhere [13]. All animal studies were conducted in accordance with the NIH and institutional guidelines on the care and use of laboratory animals. Hippocampus, trigeminal ganglion, and ovary were removed and frozen in liquid nitrogen for RNA preparation. Ovaries of five animals were fixed by immersion in Bouin fixative and were paraffin embedded. Sections cut from these blocks were processed for immunohistochemistry as indicated below.
Immunocytochemistry
Tissue sections were deparaffinized and hydrated as described [9]. Cell cultures were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde in PBS and subsequently permeabilized with saponin (0.05%). SNAP-25 was localized using a monoclonal antibody (Sternberger monoclonals Inc., Baltimore, MD; 1:500), followed by biotin-labeled secondary antibody (Camon, Wiesbaden, Germany; 1:500) and the ABC kit (Vectastain; Camon). The chromogen used was DAB. For control purposes, the first antibody was omitted or replaced by mouse normal serum.
Western Blotting
Cells and tissues were homogenized in 62.5 mM Tris-HCl buffer (pH 6.8) containing 10% sucrose and 2% SDS by sonication, mercaptoethanol was added (10%), and the samples were heated (95°C for 5 min). Fifteen micrograms of protein per lane was loaded on Tricine-SDS-polyacrylamide gels (12%), electrophoretically separated, and blotted onto nitrocellulose [14]. Blots were incubated with a monoclonal SNAP-25 antibody (Sternberger monoclonals, 1:2000) and subsequently with peroxidase-labeled secondary antibody (Camon, Wiesbaden, Germany, 1:3000). A ß-actin antibody (Sigma, 1:5000) was used to compare the protein amounts. Signals were detected with an enhanced chemiluminescence kit (Amersham Buchler, Braunschweig, Germany). For densitometric measurements, blots were digitalized using an image documentation system (MWG-biotech, Ebersberg, Germany). Integrated optical densities of the immunoreactive SNAP-25 bands were normalized to the signal obtained for actin. Results are given as mean values ± SEM.
Progesterone Assay
Culture medium from different stimulation experiments was collected and frozen at -20° C until determination of progesterone concentrations using the Serozyme-M kit from BioChem (Freiburg, Germany), as described elsewhere [9].
Reverse Transcription Polymerase Chain Reaction and Multiplex Reverse Transcription Polymerase Chain Reaction
Total RNA of cell cultures was prepared by using an RNeasy kit (Qiagen, Hilden, Germany). We used 500 ng of total RNA for reverse transcription (RT) utilizing an 18-mer polydeoxythymidine primer and Moloney murine leukemia virus reverse transcriptase (Promega, Mannheim, Germany). Polymerase chain reaction (PCR) primers for SNAP-25 were chosen to span the whole open reading frame (hu2 and hu8, Table 1). The following PCR conditions were used over 33 cycles: 5 min at 94°C of initial denaturing, 1 min at 60°C of annealing, 2 min at 72°C of extension, 15 sec at 94°C of denaturing. Taq polymerase and PCR buffer were from Promega. PCR products were subcloned into the pGEMT vector (Promega) and sequenced by a fluorescence-based didesoxy sequencing reaction on an ABI model 373A DNA sequencer (Perkin Elmer, Ueberlingen, Germany).
|
To distinguish between SNAP-25a and SNAP-25b in rat cDNA, two sense primers were designed, one corresponding to the end of exon 5a (rat5a, six mismatches for exon 5b) or the start of exon 5b (rat5b, seven mismatches for 5a), respectively. The common 3' primer was rat7. Expected product length was 282 base pairs (bp) for SNAP-25a and 365 bp for SNAP-25b. The reactions were performed in a volume of 50 µl containing 2mM MgCl2; 80 mM KCl; 16 mM Tris buffer, pH 8.3; 200 µM dNTP (each nucleotide); 2 µM each primer; 0.5 µg/µl acetylated BSA; and 0.1 U/µl Taq polymerase (Promega). PCR conditions were as follows for 35 cycles: initial denaturation at 94°C for 1 min, annealing at 55°C for 40 sec, extension at 68°C for 1 min, and denaturation at 94°C for 20 sec. To establish that SNAP-25a and b can be amplified simultaneously in one PCR tube under these conditions, we performed the following control tests: first, we included neuronal tissues known to contain different levels of SNAP-25a and b. Second, full-length rat SNAP-25a and b cDNAs in different amounts were used for further calibration (0.1 pg total cDNA per reaction).
Laser Microbeam Microdissection and Laser Pressure Catapulting
Sections (8 µm) of paraffin-embedded rat ovaries were mounted on object slides covered with a 1.35-µm polyethylene membrane (Laser Pressure Catapulting [LPC] membrane, P.A.L.M. GmbH, Bernried, Germany) and stained with hematoxylin. Single oocytes and groups of GC were dissected by laser microbeam microdissection (LMM) and transferred to a PCR tube cap by LPC using a Robot-Microbeam equipped with a N2-laser (technical advice was kindly provided by P.A.L.M. GmbH [15]). Ribonucleic acid was isolated with the Purescript kit (Gentra Systems, Minneapolis, MN). All solution volumes were reduced to one tenth of the recommended amounts. The RNA was reverse transcribed using Superscript-RT II (GibcoBRL, LifeTechnologies, Karlsruhe, Germany) and the gene-specific primer rat9 at 50°C for 1 h. Five microliters of the RT reaction were used in the first PCR. Conditions were as described above for multiplex PCR with few exceptions, namely a total volume of 25 µl, 28 cycles, and 3' primer rat8. In the second heminested PCR, 0.1 µl of the first PCR reaction was used as template; conditions were as described for multiplex PCR.
Northern Blotting
For Northern blotting, 10 µg total RNA was denatured at 65°C in a formamide-formaldehyde-MOPS buffer, loaded onto a 1.1% agarose gel containing 2.2 M formaldehyde, and blotted onto a nylon membrane (Hybond-N; Amersham) using 20x SSC and a vacuum blotter (Appligene Oncor, Heidelberg, Germany) as described elsewhere [2]. After linearization (NotI or NcoI), SNAP-25a and ß-actin clones were used as templates for in vitro transcription using 32P-UTP and T7- or SP6-RNA-polymerase (Promega). Transcripts were purified with Nick columns (Pharmacia) and hybridized at 60°C overnight. Subsequently, blots were washed five times at 65°C in 0.1x SSC and 0.1% SDS and dried. Autoradiograms were developed after 1 to 5 days. Densitometric analyses were performed as described for Western blots.
Statistical Analyses
One-way ANOVA for repeated measures was used to analyze the expression level after various stimulations. Dunnett's multiple comparison test was used as a post hoc test if significant different means were detected. Progesterone assay was analyzed by the one-sample t-test. Results are expressed as mean ± SEM. A P < 0.05 was considered significant.
RESULTS
Distribution of SNAP-25
As expected, nerve fibers of rat and human ovaries were stained using a monoclonal antibody directed against SNAP-25. Unexpectedly however, nonneuronal structures also showed specific SNAP-25 immunostaining. Oocytes in follicles of all developmental stages displayed strong immunoreactivity, in rat as well as in human ovary (Fig. 1). In addition, steroid-producing GC and luteal cells were positive in both species. SNAP-25 staining of GC was most conspicuous in GC of growing antral follicles, although not all GC of these follicles were stained (Fig. 1F). This staining pattern may be due to functional differences between GC that have been described for cumulus and mural GC [16, 17]. Thecal cells were immunopositive in rat ovary, but human thecal cells were negative in all samples investigated (n = 3). Besides this difference, the comparison of the immunohistochemical data obtained in rat and human revealed a very similar pattern.
|
To further investigate SNAP-25 expression in GC, we used two in vitro systems, the rat GFSHR-17 cell line expressing FSH receptors and human luteinized GC expressing LH/hCG receptors. Either cell type was immunopositive for SNAP-25 like their counterparts in the ovary, GC of antral follicles and luteal cells. The GFSHR-17 cells showed membrane-associated and intracellular staining, whereas in human GC, a perinuclear and occasionally a granular staining was present (Fig. 2).
|
Analysis of SNAP-25 and Its Isoforms
We performed RT-PCR experiments using primers spanning the whole open reading frame of SNAP-25 (hu2 and hu8, Table 1). This primer set amplified products of the expected size of 621 bp from human GC, GFSHR-17 cells, or rat ovary despite up to two mismatches for the rat template. Sequence analysis of five independent clones derived from human GC showed the expression of the SNAP-25a isoform. However, in rat ovary, we found both SNAP-25 isoforms a and b (two independent clones of each isoform). These data raised the possibility of cell type-specific isoform expression. Alternative splicing of two variants of exon 5 results in SNAP-25a or b [18]. Exons 5a and 5b have identical length and are 73% homologous. Because of their close similarity, the two isoforms cannot readily be distinguished by antibodies, mRNA length, or RNA probes. We therefore used a PCR approach with two sense primers, one homologous to the start of exon 5b (rat5b), the other homologous to the end of exon 5a (rat5a), together with a common antisense primer (rat7; see Fig. 3). These primers allowed to amplify SNAP-25a and b in one test tube. We tested the specificity of this approach and calibrated it using SNAP-25a or b DNA and mixtures thereof as templates. Reverse-transcribed RNA from hippocampus and trigeminal ganglion were used as positive tissue controls. Adult hippocampus is known to express predominantly SNAP-25b and, at a lower level, also SNAP-25a [19, 20]. This is consistent with our results, which show the predominant expression of SNAP-25b. Exclusively SNAP-25b occurred in the trigeminal ganglion (Fig. 3). This validated PCR approach was then applied to GFSHR-17 cells. These cells were shown to express SNAP-25a (Fig. 3).
|
To investigate the cell type-specific expression pattern in the ovary, we used hematoxylin-stained paraffin sections of rat ovary. Single oocytes and groups of GC were microdissected with a laser microbeam. The dissected parts were moved to a PCR tube cap by laser pressure catapulting (Fig. 4) [15, 21]. Heminested RT-PCR revealed the exclusive expression of SNAP-25a in GC in three independent experiments. In contrast, oocytes expressed SNAP-25b only (two independent experiments; Fig. 4), the isoform predominating in the hippocampus and other parts of the adult brain.
|
Regulation of SNAP-25 Expression In Vitro
Functional differentiation of GC during follicular development is controlled by FSH, whereas luteal cells are regulated by LH/hCG [17, 22, 23]. We investigated the influence of gonadotropins on SNAP-25 expression. The rat granulosa cell line GFSHR-17 is known to express functional FSH-receptors [12]. FSH stimulation induced cell rounding and aggregation (data not shown) as described for primary cultures of rat GC [24, 25], indicating close similarities between GFSHR-17 cells and primary cultures of immature rat GC.
Unstimulated cells showed a SNAP-25 signal in Northern blots that was strongly increased by stimulation with FSH for 24 h (Fig. 5). This effect could be mimicked by stimulating cells with the stable second-messenger analogue db-cAMP. Both stimulations could be blocked by the PKA-inhibitor H-89 [26]. To examine whether these mRNA changes are paralleled at the protein levels, Western blots were probed with a monoclonal SNAP-25 antibody. Results revealed very similar changes in the SNAP-25 levels in response to the stimulations (Fig. 5). Statistical analyses of densitometric values revealed a significant increase for FSH (5.1-fold ± 1.8) and db-cAMP treatment (4.5 ± 1.2, n = 5). The PKA inhibitor H89 prevented this increase of SNAP-25 level (Fig. 5). These results suggest that SNAP-25 is subjected to transcriptional regulation by FSH via a cAMP- and PKA-dependent pathway.
|
To study the role of LH/hCG on SNAP-25, luteinized GC derived from IVF patients were used. These cells are known to express LH receptors and responded to stimulation by hCG (10 IU/ml) by a significant increase in progesterone secretion after 24 and 48 h (Fig. 6C). In contrast, Northern blot analysis showed SNAP-25 transcripts of 2.1 kb in control and stimulated cells, which, upon densitometric analysis, were not different. Likewise, the densities of SNAP-25 bands in Western blots were unchanged (Fig. 6).
|
DISCUSSION
The synaptosome-associated, 25-kDa protein was initially described in the nervous system [3], and its importance for the mechanism of regulated exocytosis was elucidated by the discovery that SNAP-25 is the target of botulinum neurotoxins A and E. These neurotoxins proteolytically cleave SNAP-25 and thus inhibit neurotransmitter release [27, 28]. Neuroendocrine cells, sharing their neuroectodermal origin with neurons, were also found to express SNAP-25. Its cleavage by botulinum neurotoxins likewise causes inhibition of exocytosis of hormones [5–8]. Our results show that SNAP-25 is present unexpectedly in nonneuronal and nonneuroendocrine compartments of the ovary. Thus, endocrine cells involved in steroid production, such as follicular cells and cells of the corpus luteum, express SNAP-25, and it is also present in oocytes.
To our knowledge, the presence of SNAP-25 in mammalian oocytes has not been reported before. However, SNAP-25 is present in sea urchin eggs and was shown to be involved in cortical granule exocytosis [29–31]. This process is important for avoiding polyspermy during fertilization of almost all vertebrates and many invertebrates. It is plausible that in human and rat oocytes, SNAP-25 may be functionally involved in the same process. However, we found SNAP-25 to be present in all oocytes, including those of preantral follicles, long before ovulation. This suggests its involvement in other processes. For example, primate oocytes are a source of catecholamines because they are able to take up dopamine and to synthesize and release norepinephrine [1]. Exocytosis of catecholamines in chromaffin cells is mediated by SNARE proteins, including SNAP-25 [6, 32]. Thus, the release of catecholamines from mammalian oocytes may be a process mediated by these proteins.
Reverse transcription PCR of RNA of rat oocytes isolated by laser capture microdissection revealed the expression of SNAP-25b, one of the two isoforms of SNAP-25; these isoforms are the result of alternative splicing processes between two versions of exon 5. Both versions contain four conserved cysteine residues subjected to posttranslational fatty acylation [18, 33]. Mutational analysis demonstrated that the palmitoylation pattern is crucially involved in the subcellular distribution of the isoforms [34–36]. Nerve growth factor is known to induce sprouting of processes in neuroendocrine PC12 cells. Using this model, transfection studies with SNAP-25a and b showed a differential distribution of the isoforms. The a isoform of SNAP-25 was distributed throughout the cell, but SNAP-25b was present in branching points and tips of processes [19]. This implies an isoform-specific protein sorting to specific membrane domains, suggesting an involvement of the isoforms in constitutive and regulated exocytotic processes, respectively.
Isoform-specific functions are also suggested by the developmental expression. SNAP-25a is the prevalent isoform in the embryonic nervous system, whereas SNAP-25b predominates in the adult brain [19, 20]. This also suggests the involvement of SNAP-25b in regulated exocytosis of neurotransmitters in the mature nervous system, whereas SNAP-25a plays a role in constitutive exocytosis required for growth of neuronal processes during development. Direct evidence for the participation of SNAP-25 in growth of neuronal processes was provided by inhibiting SNAP-25 expression with antisense oligonucleotides which caused a decrease in axonal growth [37]. Moreover, cleavage of SNAP-25 by botulinum neurotoxin A inhibited both axonal and dendritic growth [38]. Kainate-induced CNS injury, followed by regenerative processes and plastic changes, caused a selective up-regulation of SNAP-25a [20, 39] suggesting the participation of this isoform in growth processes and constitutive exocytosis in the adult brain. Taken together, the expression of SNAP-25b in oocytes hints at its involvement in regulated exocytotic processes rather than in constitutive exocytotic events.
The immunohistochemical detection of SNAP-25 in theca cells, GC, and cells of the corpus luteum was surprising. Although a major task of these cells is the secretion of steroids, GCs particularly are known to synthesize and secrete a plethora of different products. Besides steroid hormones and prostaglandines, they produce a wide variety of peptides and growth factors, such as basic fibroblast growth factor, insulin-like growth factor I and II, epidermal growth factor, leukemia inhibitory factor, transforming growth factor- and -ß, tumor necrosis factor-, vascular endothelial growth factor, cytokines (interleukin-1 and -6), and glycoprotein and peptide hormones (activin and inhibin, follistatin, oxytocin, and leptin; for references, see [40]). Some of these products may be released constitutively, others by regulated exocytosis, or, in the case of steroids, by diffusion through the plasma membrane. In addition, cumulus cells and adjacent GCs in the ovarian follicles have been shown to secrete several growth factors by an apocrine-like mechanism [40]. Despite the wide variety of products released by different mechanisms, GCs express the isoform SNAP-25a. In this respect, GC are similar to neuroendocrine cells, which also express predominantly SNAP-25a to secrete their hormones [19, 41].
It is well established that biosynthesis and secretion of products of ovarian endocrine cells are stimulated by FSH and LH/hCG [42]. This includes estrogen and progesterone secretion, as well as follistatin and glucosaminoglycans [43, 44]. If SNAP-25a is involved in the secretion of these products, one may anticipate up-regulation of SNAP-25 by gonadotropins. We found in FSH-responsive GFSHR-17 cells that FSH caused a robust increase in SNAP-25. This FSH effect was mediated by the cAMP-protein kinase A (PKA) transduction pathway. Thus, these in vitro results reflect the low or absent SNAP-25 observed in small follicles, which are in contrast to strong SNAP-25 expression in GC of large antral follicles (this study).
Little is known regarding the regulation of SNAP-25. In neurons, phosphorylation of SNAP-25 by Ca2+/calmodulin kinase II is a possible mechanism of posttranslational regulation that could contribute to the tuning of the exocytotic apparatus and the regulation of the efficacy of synaptic transmission [45]. Evidence for transcriptional regulation of SNAP-25 has been provided only in a few previous studies. Thus estrogen reduced pituitary SNAP-25 mRNA levels in ovariectomized rats, whereas the expression of other proteins involved in hormone exocytosis remained unchanged [46]. A db-cAMP-induced increase in the SNAP-25 level was reported to occur in PC12 cells [47]. However, it is unknown whether activation of receptors leading to cAMP production would cause similar effects. Likewise, arachidonic acid and calcium can moderately increase SNAP-25 expression in these cells and hippocampal explants but not in cerebellar explants [48]. A decrease of SNAP-25 in neuronal cells is a consequence of various pathologic stimuli or cellular lesions [49, 50]. Besides the experimental studies mentioned, to our knowledge, no reports on the regulation of SNAP-25 under physiological conditions have been published.
Our results show for the first time that SNAP-25 is present in mammalian oocytes and endocrine ovarian cells. SNAP-25a and b isoforms are differentially expressed suggesting their involvement in the specific functions of these cell types. Moreover, our results obtained in granulosa cells indicate that FSH is a physiological regulator of SNAP-25.
ACKNOWLEDGMENTS
We thank Barbara Zschiesche, Gerhard Prechtner, and Andreas Mauermayer for their excellent technical assistance.
FOOTNOTES
First decision: 2 November 1999.
1 Support for this study was provided by the Volkwagen-Stiftung Fonds der Chemischen Industrie and the DFG (Ma1080/12-1). A.A. is the incumbent of the Joyce and Ben B. Eisenberg Professorial Chair of Molecular Endocrinology and Cancer Research. 
2 Correspondence: M. Gratzl, Anatomisches Institut, Technische Universität München, Biedersteiner Str. 29, 80802 München, Germany. FAX: 49 89 397035; gratzl{at}lrz.tu-muenchen.de
Accepted: April 10, 2000.
Received: October 11, 1999.
REFERENCES
-SNAP interaction with the core complex. Proc Natl Acad Sci U S A 1997; 93:11945–11949.This article has been cited by other articles:
|
|
 |
|
  M. Greiner, A. Paredes, V. Rey-Ares, S. Saller, A. Mayerhofer, and H. E. Lara Catecholamine Uptake, Storage, and Regulated Release by Ovarian Granulosa Cells Endocrinology, October 1, 2008; 149(10): 4988 - 4996. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shimada, Y. Yanai, T. Okazaki, Y. Yamashita, V. Sriraman, M. C. Wilson, and J. S. Richards Synaptosomal-Associated Protein 25 Gene Expression Is Hormonally Regulated during Ovulation and Is Involved in Cytokine/Chemokine Exocytosis from Granulosa Cells Mol. Endocrinol., October 1, 2007; 21(10): 2487 - 2502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Jo, M. C. Gieske, C. E. Payne, S. E. Wheeler-Price, J. B. Gieske, I. V. Ignatius, T. E. Curry Jr., and C. Ko Development and Application of a Rat Ovarian Gene Expression Database Endocrinology, November 1, 2004; 145(11): 5384 - 5396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. SASSON, A. DANTES, K. TAJIMA, and A. AMSTERDAM Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action FASEB J, July 1, 2003; 17(10): 1256 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fritz, I. Wessler, R. Breitling, W. Rossmanith, S. R. Ojeda, G. A. Dissen, A. Amsterdam, and A. Mayerhofer Expression of Muscarinic Receptor Types in the Primate Ovary and Evidence for Nonneuronal Acetylcholine Synthesis J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 349 - 354. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
