| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317
ABSTRACT
During the periovulatory period, the mammalian ovary is the site of dramatic functional and structural changes, leading to oocyte maturation, follicle rupture, and corpus luteum formation. To a large extent, these processes result from changes in the transcriptome of various ovarian cell types. To develop a broader view of periovulatory changes in gene expression in the ovary and to identify further genes involved in periovulatory events, we used the recently developed DNA array technology. Immature female eCG-primed rats were killed either immediately before or 6 h after ovulation induction with hCG. Total ovarian RNA was isolated and used to prepare radiolabeled cDNA probes, which were hybridized to DNA arrays representing approximately 600 rat genes. Quantitative analysis identified a multitude of regulated gene messages, including several genes involved in extracellular matrix degradation and lipid/steroid metabolism previously reported to be induced by hCG. This screening also identified a group of candidate genes whose ovarian expression and gonadotropin regulation was hitherto unknown. The induction of three of these genes, encoding cutaneous fatty acid-binding protein, the interleukin-4 receptor alpha chain, and prepronociceptin, was confirmed and further characterized by Northern blot analysis. In addition, in situ hybridization analysis showed that hCG administration resulted in exclusive or predominant expression of all three genes in theca cells. These results demonstrate that DNA arrays can be used to identify genes regulated during the periovulatory period, thus contributing to a more detailed understanding of the molecular mechanisms of ovulation.
corpus luteum function, granulosa cells, luteinizing hormone, ovary, ovulation, steroid hormones, theca cells
INTRODUCTION
During the periovulatory period, profound changes take place in the ovary, culminating in oocyte maturation, follicle rupture, and corpus luteum formation. These functional and structural changes are largely dependent on the regulation of mRNA expression of key genes involved in these processes.
Over the past decades, various approaches including Northern blot hybridization, differential display, and subtractive hybridization have been used to identify a number of gene messages regulated during the periovulatory period [1–5]. Recent advances in the genome projects of humans and other species and the development of microarray analysis tools [6, 7] now provide the opportunity to simultaneously analyze the expression patterns of hundreds to thousands of genes and will ultimately allow us to achieve a global view of gene expression.
Data from Unigene database searches and serial analysis of gene expression polymerase chain reaction results suggest that a large proportion of all the genes in the human genome are expressed in the ovary [8–10]. It is also becoming apparent that many genes originally identified in nonovarian tissues are also expressed within the ovary, where they serve important functions. Here, we report the use of membrane-based DNA arrays (from Clontech Laboratories, Palo Alto, CA) to identify the induction or repression of known genes during the first 6 h following hCG induction of ovulation in the ovaries of gonadotropin-primed immature rats. Using this approach, we confirmed the expression pattern of several genes known to be regulated during the preovulatory period. We also identified a number of additional genes previously studied only in nonovarian tissues. The ovarian expression and gonadotropin induction of three of these genes, those encoding the cytosolic protein cutaneous fatty acid-binding protein (C-FABP), the plasma membrane receptor for interleukin-4 (IL-4), and the precursor for the extracellular ligand nociceptin, were confirmed and further characterized by Northern blot and in situ hybridizations.
MATERIALS AND METHODS
Animal Treatment
Immature female Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) with a weight range of 50–60 g on Day 24 of age were housed under 12L:12D lighting conditions (lights-on at 0700 h). At 0900–1000 h on Day 25 of age, the animals were primed with 10 IU eCG (Calbiochem, La Jolla, CA) s.c. In addition, the rats received two s.c. injections of the GnRH antagonist Org 30850 (40 µg/kg body weight; Organon, Oss, The Netherlands) at 0900–1000 h on Days 25 and 26 of age to suppress endogenous pituitary gonadotropin secretion and prevent spontaneous ovulation [11]. After 48 h of eCG priming, rats were killed either immediately (control group) or at 6 h after an i.p. injection with 10 IU hCG (hCG group) (Schein Pharmaceuticals, Florham Park, NJ), and the ovaries were collected for RNA extraction. Additional animals were observed for more than 12 h after hCG administration to verify ovulation by checking for the presence of oocytes in the oviducts. For time course analyses of mRNA expression and for in situ hybridization studies, rats treated similarly (but not subjected to a GnRH antagonist) were killed at different time points after hCG injection, and the ovaries were collected for RNA extraction or fixed, respectively. All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University, and experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.
RNA Extraction and DNA Array Hybridization
Ovaries were dissected free of adherent tissue, snap-frozen in a dry ice-ethanol bath, and stored at -70°C. Total RNA from whole ovaries was extracted using TRIzol reagent (Gibco BRL, Gaithersburg, MD) and the Atlas Pure RNA Extraction Kit (Clontech). Radiolabeled complex cDNA probes were reverse transcribed from isolated ovarian RNA (five rats/group) and hybridized to separate Atlas Rat cDNA Expression Arrays (Clontech) following the manufacturer's instructions. The membranes were then exposed to PhosphorImager screens for 3 wk and scanned on a Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). Quantification of PhosphorImager signals was performed on an image analyzer system (Imaging Research, St. Catherines, ON, Canada).
Generation of Specific cDNA Probes for Northern Blot Analysis
The cDNA sequences for the genes of interest were used to search the NCBI expressed sequence tag (EST) database for rodent ESTs. To prevent nonspecific cross-hybridization with mRNAs possessing paralogous open reading frames, only clones with inserts corresponding to 3' untranslated regions of the respective cDNAs were selected (Research Genetics, Huntsville, AL). The correct identity of the inserts was verified by DNA sequencing, and inserts were released from their cloning sites in the pT7T3D-Pac vectors using the restriction enzymes NotI/EcoRI. Specific cDNA probes for ovarian genes of interest and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 32P radiolabeled by random priming using a commercial kit (Gibco).
Northern Blot Analysis and Quantification of Message Levels
Twenty micrograms of total ovarian RNA per lane were run on agarose-formaldehyde gels before transfer to nitrocellulose membranes and cross-linking with ultraviolet light. Northern blots were prehybridized and then hybridized to the respective 32P-radiolabeled cDNA probes using ExpressHyb solution (Clontech) according to the manufacturer's instructions. Following several washes in 0.1x standard saline citrate (SSC), 0.5% SDS at 57–60°C, membranes were exposed to film (at -70°C) or to PhosphorImager screens.
Following exposure of membranes to PhosphorImager screens, the signal intensity for each transcript of interest was quantified using a Storm 860 PhosphorImager and ImageQuant image analysis software (Molecular Dynamics). The intensity values for the transcripts in RNA samples extracted from rat ovaries at 0, 3, 6, 9, 12, and 24 h after hCG injection were then normalized to the respective GAPDH signal (from a subsequent control hybridization). The results are expressed as fold induction divided by the signal at 0 h, which was arbitrarily set as 1. Results are presented as mean ± SEM of samples from three different animals at each time point.
In Situ Hybridization Studies
Ovaries were fixed at 4°C for 6 h in 4% paraformaldehyde in PBS, followed by immersion in 0.5 M sucrose in PBS overnight. Cryostat sections (7 µm thick) were mounted on microscope slides coated with poly-L-lysine (Sigma, St. Louis, MO), fixed in 4% paraformaldehyde in PBS, and stored at -80°C until analyzed. The hybridization procedure was essentially the same as previously described [12]. Sections were pretreated serially with 0.2 M HCl, 2x SSC, pronase E (0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in triethanolamine and dehydrated in ascending grades of ethanol. The antisense and sense probes were labeled with [35S]UTP (1000 Ci/mmol; Amersham, Piscataway, NJ). The sections were hybridized overnight at 45°C in 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1x Denhardt solution, 10% dextran sulfate, 1 µg/ml carrier tRNA, and 10 mM dithiothreitol. Following ribonuclease A (20 µg/ml) treatment at 37°C for 30 min, posthybridization washing was performed to a final stringency of 0.1x SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and exposed at 4°C for 1 wk before development. The slides were stained with hematoxylin and eosin and mounted with DPX Mountant (Electron Microscopy Sciences, Ft. Washington, PA). The slides were photographed using a Zeiss 35-mm camera and microscope (MC80DC; Carl Zeiss, Oberkochen, Germany) with bright- and darkfield illumination. To allow for direct comparison of the ovarian sections from different experimental groups, all slides were processed simultaneously and under identical conditions.
RESULTS
DNA Array Hybridization
Ovarian RNA from eCG-primed rats killed at 0 h and 6 h following i.p. administration of 10 IU hCG was used to generate radiolabeled complex cDNA probes. These probes were then separately hybridized to membrane-based cDNA arrays representing 597 known rat genes. Because each gene was spotted twice onto each of the arrays, a total of 2388 data points were quantified using a PhosphorImager and an image analysis system. The induction ratio for each of the 597 genes was determined by calculating the ratio of signal detected at 6 h versus that at 0 h following hCG treatment. For genes with a decrease in mRNA expression after hCG administration, the repression ratio (reciprocal value of the induction ratio) is given to facilitate comparison.
All changes in gene expression, as determined by the DNA array hybridizations, fall into a range between the maximal repression ratio of 1.83 and the maximal induction ratio of 4.04 (Fig. 1). However, the vast majority of the 597 messages studied showed only small changes in expression between the 0 h and 6 h samples, with almost two thirds of mRNAs (65.6%) having induction or repression ratios of up to 1.15 and more than three quarters (77.9%) having induction or repression ratios of up to 1.25. Our further analysis therefore focused on the messages with the highest induction or repression ratios, indicating their regulation by hCG in the preovulatory ovary.
|
Cutaneous Fatty Acid-Binding Protein
The gene encoding C-FABP, with an induction ratio of 4.04, had the highest preovulatory upregulation among the 597 messages quantified on the arrays (Table 1). Northern blot studies confirmed the dramatic increase in ovarian C-FABP mRNA, showing a 7.8-fold induction (±0.68; P < 0.05) at 6 h after hCG administration, which remained at this level up to the time of ovulation and subsequently dropped to near baseline levels by 24 h following hCG injection (Fig. 2, A and B). In situ hybridization studies demonstrated only negligible expression of C-FABP mRNA before hCG treatment (data not shown) compared with a strong expression localizing to the theca layer of antral and preovulatory follicles 6 h thereafter (Fig. 3). The message with the highest repression identified in our DNA array experiments encodes another fatty acid-binding protein, heart fatty acid-binding protein (H-FABP) (Table 2).
|
|
|
|
IL-4 Receptor
On the arrays, the IL-4 receptor 
chain (IL-4R) message showed a 2.67-fold induction 6 h after hCG induction of ovulation, making it the second most highly induced mRNA in this study (Table 1). In Northern blot experiments, the IL-4R mRNA reached its maximum (3.3-fold ± 0.47-fold induction; P < 0.05) at 3 h after hCG administration, followed by a gradual decline to baseline levels after 24 h (Fig. 2, C and D). In situ hybridization studies showed minimal baseline expression throughout the ovary (data not shown). Following hCG administration, there was marked upregulation of the IL-4R mRNA in the theca cell layer of antral and preovulatory follicles, with a lesser induction in the granulosa and interstitial cell layer (Fig. 4).
|
Prepronociceptin
With a 2.24-fold increase, the prepronociceptin (PNOC) message had the fourth highest level of induction in the DNA array study (Table 1). In Northern blot experiments, the induction of PNOC exceeded the induction values of both C-FABP and IL4R; there was a 10.9-fold increase by 6 h following hCG induction of ovulation, reaching a 13.3-fold (±2.47; P < 0.05) increase over the original levels by 9 h, before decreasing during the postovulatory period (Fig. 2, E and F). In situ hybridization studies showed a marked upregulation of PNOC mRNA in the theca cell layer of antral and preovulatory follicles at 6 h after hCG administration (Fig. 5).
|
DISCUSSION
The periovulatory period is characterized by a host of changes in ovarian gene expression leading to follicle rupture, oocyte maturation, and formation of the corpus luteum. Although some of the differentially expressed messages found in this study already have known functions in the context of ovulation, the identification of other up- or downregulated messages provides a starting point for further investigations.
Cutaneous Fatty Acid-Binding Protein
As demonstrated in the DNA array study, Northern analysis, and in situ hybridizations, the ovarian C-FABP mRNA is strongly induced following hCG induction of ovulation (mainly in the theca cells of large follicles). By contrast, the message for H-FABP was the most highly repressed message among the 597 genes represented in the array.
The fatty acid-binding proteins (FABPs) are among the most abundant cytoplasmic constituents of parenchymal cells [13]. These small cytosolic proteins enhance the intracellular transport and metabolism of fatty acids and may show an affinity for other ligands, such as retinoic acid, bile acids, or prostaglandins. Different genes encode at least eight types of FABPs, which differ in their specific binding properties and have each been named after the tissue of major occurrence. Although this study is the first to show expression and regulation of C-FABP in the ovary, H-FABP expression in theca cells of the rat ovary (but not its preovulatory regulation) has been described previously [14]. Three other genes for FABPs—intestinal FABP, adipocyte FABP, and liver FABP—were also represented on the array, but the quantification revealed no clear regulation of the mRNA by hCG (data not shown).
Expression of the C-FABP and H-FABP genes in theca cells of the rat ovary and their contrary regulation by hCG suggest a differential role for these two FABPs in the preovulatory ovary, possibly relating to the changes in lipid metabolism accompanying corpus luteum formation. Further studies will be needed to address the function of this group of proteins in the ovary.
IL-4 Receptor
This study is the first to show preovulatory induction of the IL-4R message in the ovary. IL-4 is a cytokine produced by subsets of T cells, basophils, and mast cells that regulates proliferation and differentiation of a variety of cells. Its signalling is mediated by the heterodimeric IL-4 receptor, a member of the hematopoietin receptor superfamily expressed on lymphocytes and on a range of nonimmune cells [15]. IL-4 binds with high affinity and specificity to the IL-4 receptor chain (IL-4R), which subsequently recruits a second chain, usually the gamma common chain, to initiate transmembrane signalling.
So far, there have been few observations indicating a possible involvement of IL-4 or its receptor in periovulatory events. A recent study showed that IL-4 promotes progesterone production by human luteal cells in culture [16]. In addition, IL-4 has an important role in regulating inflammatory responses [15], which have been proposed to bear similarity to the ovulatory process [3]. Other interleukins, such as IL-1 beta, IL-6, and IL-8, and their respective receptors have been suggested to act as intermediaries in the ovulatory process in rodents and humans [17–20]. Viable IL-4R-deficient mice have been generated; however, their reproductive phenotype has not been reported [21]. Our findings suggest that the IL-4 system, analogous to other interleukin systems, could serve as a mediator for periovulatory changes in the ovary.
Prepronociceptin
Of the three genes studied by Northern analysis, PNOC showed the highest ovarian mRNA induction in response to ovulatory hCG stimulus. PNOC is the precursor of nociceptin (also referred to as orphanin FQ) and nocistatin, two neuropeptides involved in pain transmission in the central nervous system [22, 23]. The 17-amino-acid nociceptin is the natural agonist of the opioid receptor-like-1 receptor, a G protein-coupled receptor. The organization and structure of the PNOC gene closely resemble the genes encoding the opioid peptide precursors preproenkephalin, preprodynorphin, and preproopiomelanocortin (POMC). The ovary appears to be the only organ outside of the central nervous system expressing the PNOC gene [22], but the intraovarian expression and function of this gene have not been addressed previously.
The mRNA for the closely related POMC gene is expressed in follicles and corpora lutea of the rat ovary [24], and peptides derived from this gene are found in high concentrations in the follicular fluid of preovulatory human follicles [25]. Our findings for the first time implicate nociceptin and/or nocistatin as possible paracrine factors in the periovulatory ovary. However, knockout mice in which the genes for nociceptin or its receptor have been disrupted are healthy and fertile [26, 27], suggesting that loss of these gene products can be compensated for to preserve essential reproductive functions.
Extracellular Proteases and Their Inhibitors
Follicle rupture as a key component of the ovulatory process is the consequence of changes in composition and structure of the follicular wall brought about by the local interplay of extracellular proteases and their inhibitors [4]. In our study, plasminogen activator inhibitor 1 was identified among the 10 most highly induced messages represented on the array (Table 1), consistent with previous descriptions of its induction by hCG in the granulosa (and theca) cells of preovulatory follicles in the rat [28, 29]. Likewise, the message for tissue inhibitor of metalloproteinase 1 (TIMP-1) was found to belong to the 5% of most highly induced genes on the array, in accordance with earlier studies on its expression following ovulation induction in the rat [29, 30]. The genes for TIMP-1, matrix metalloproteinase (MMP) 19 [5], and the extracellular metalloproteinase ADAMTS-1 [31] have been recently proposed as possible targets of the immediate-early transcription factor Egr-1, which is also strongly upregulated within a few hours of hCG induction of ovulation in the rat ovary [32]. Also identified among the top 5% of upregulated messages were the mRNAs for MMP-2 (gelatinase A) and urokinase-type plasminogen activator receptor (Table 1), both of which are known to be induced by hCG in the preovulatory rat ovary [33, 34].
Steroid and Lipid Metabolism
Following the induction of ovulation by LH/hCG, a series of morphological and biochemical changes initiate the eventual conversion of ruptured follicles into corpora lutea. This process is accompanied by the increased expression of proteins involved in lipid uptake and metabolism and by a specific set of steroidogenic enzymes.
One of the 10 most highly induced messages identified in this study is the one for scavenger receptor class B type I (SR-BI), a cell surface lipoprotein receptor that mediates the selective uptake of high-density lipoprotein cholesterol as a substrate for steroid hormone production (Table 1). This finding implies that the high levels of SR-BI gene expression observed in the rat corpus luteum [35] result from a message induction already beginning in the preovulatory period.
Also featured among the 10 most highly induced messages in the array is the mRNA encoding the low-density lipoprotein receptor (LDL receptor) precursor (Table 1). Although the preovulatory induction of LDL receptor mRNA has not been described in the rat, its upregulation by an ovulatory hCG stimulus in granulosa cells has been described in the macaque [36].
The DNA arrays used in this study contained only a subset of the genes encoding steroidogenic enzymes, but both 20-alpha-hydroxysteroid dehydrogenase (20-HSD) and cholesterol side-chain cleavage cytochrome P450 (P450scc) were among the 5% of most highly upregulated messages (Table 1). Ovarian gene expression for P450scc, which catalyzes the rate-limiting step in the conversion of cholesterol to pregnenolone, is known to increase following hCG induction in the rat, reaching its maximum after ovulation [37]. The induction of 20-HSD message detected in our array experiments is likely to be the basis for the reported increase in catalytic activity of this enzyme during the preovulatory period [38].
Receptors and Signal Transduction Molecules
Consistent with earlier observations, both the LH receptor mRNA and the message for cAMP-dependent protein kinase type IIß regulatory chain (Table 2) were among the most highly repressed genes [39, 40]. Moreover, the estrogen receptor-beta mRNA was among the messages most repressed by hCG in the preovulatory ovary (Table 2), an effect that has been described elsewhere [41].
Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT family of proteins and ranked fifth in its induction by hCG on the arrays (Table 1). Following its rapid phosphorylation in response to various cytokines and growth factors, including IL-6, epidermal growth factor, or leptin, it acts as a transcription factor for acute phase response genes. An increase of STAT3 protein levels in rat granulosa cells following induction of luteinization by hCG has recently been described [42].
Also identified among the 10 most highly induced messages is the one for inhibitor of DNA binding 1 (ID1), a protein that forms inactive heterodimers with basic helix-loop-helix transcription factors and thereby interfering with their DNA binding. ID1-deficient mice appear indistinguishable from wild-type mice, whereas homozygous disruption of both ID1 and the related ID3 genes results in an embryonically lethal phenotype, apparently because of a defect in angiogenesis [43]. The expression, regulation, and function of ID1 in the ovary are currently unknown, but it is tempting to speculate that the preovulatory induction of ID1 might control angiogenic processes associated with luteinization.
Other Genes
The third highest message induction within 6 h of hCG administration was measured for the gene encoding ornithine decarboxylase (ODC). The stimulation of ODC message and activity by hCG in the preovulatory rat ovary is a well-known phenomenon that has been studied extensively [44, 45]. ODC catalyzes the conversion of ornithine to putrescine, which is the first (and apparently rate limiting) step in polyamine biosynthesis. Elevated levels of polyamines (putrescine, spermidine, and spermine) appear to stimulate cell proliferation [46].
DNA topoisomerase II alpha (TOP2A), the mRNA for which is induced 6 h following hCG administration (Table 1), belongs to a group of enzymes that catalyze changes in the topological states of DNA. A specific role for topoisomerase II in the ovary is currently not known.
A number of other ovarian messages were repressed following hCG treatment of eCG-primed rats (Table 2). Many of these genes, however, belong to larger gene families that currently have no defined role in ovarian physiology. The comparative lack of genes known to be repressed by hCG could be due to a predominance of gene induction over repression events but might also reflect a bias of researchers towards the identification and study of upregulated rather than downregulated mRNAs.
Advantages and Limitations of Array Analysis
In this study, we showed that DNA array screenings provide a valid alternative to the established differential display approach [31, 32] for the identification of genes with periovulatory induction in the ovary. Advantages of the DNA array technique include the large number of genes that can be surveyed in a single experiment and the straightforward identification of regulated genes from their position in the array. Although the insights to be gained from DNA array studies are limited to the genes (or in other cases the EST clones) represented on the membrane or glass chip, this disadvantage is likely to diminish as larger arrays become available.
In our array experiments, we used RNA isolated from whole ovaries to gain an overview of the regulation of almost 600 genes in the ovary. As a consequence of this experimental design, the study was prone to missing increases or decreases of messages limited to any small compartment within the ovary (e.g., oocytes of preovulatory follicles) because these effects would likely be rendered undetectable or counterbalanced by changes in other cell types or parts of the ovary. However, the induction or repression ratios given above are likely to underestimate the quantitative changes occurring within the group of cells actually expressing the respective message because these cells may constitute only a small fraction of the entire ovarian tissue.
From the array studies and Northern blot hybridizations, it is impossible to determine whether the "induction" or "repression" seen for an individual mRNA species is due to changes in the rate of transcription or alterations in message stability. In this study, the ovarian mRNA localization for the three genes studied in detail has been individually determined through in situ hybridization experiments. Future studies comparing gene expression profiles of the ovary as a whole with those of specific cells or structures therein (e.g., granulosa cells, preovulatory follicles) will add a topographical dimension to the description of the ovarian transcriptome.
For C-FABP, IL-4R, and PNOC, the induction ratios determined by DNA array hybridization underestimated the ones obtained from Northern blots. For example, a 2.24-fold induction measured on the array corresponds to a 10.9-fold induction at the same time point determined using Northern blot hybridizations. These differences have been observed previously and may result from the different hybridization kinetics underlying the two technologies [7]. The induction effects studied here at the mRNA level need to be verified at the protein and functional levels before definite conclusions about an involvement of these gene products in periovulatory events can be drawn.
In summary, we demonstrated the potential of DNA arrays to broaden our understanding of periovulatory gene regulation. The identification of induced/repressed genes, some of which already have well-characterized functions and pathways outside the ovary, can help researchers generate new hypotheses about and insights into ovarian physiology. The constantly evolving DNA array technology will therefore become an indispensable tool in ovarian research as in other fields of biomedicine.
ACKNOWLEDGMENTS
We are grateful to Michael Makhanov and Li Zhu at Clontech Laboratories (Palo Alto, CA) for their help with quantitative analysis of the DNA array results, to Jessica Kelly for assistance with animal treatments and tissue collection, and to Lenus Kloosterboar from Organon (Oss, The Netherlands) for providing the GnRH antagonist Org 30850.
FOOTNOTES
First decision: 26 December 2000.
1 This study was supported by NICHD/NIH through a cooperative agreement (U54-HD31398 to A.J.W.H.) as part of the Specialized Cooperative Centers Program in Reproduction Research, by the Core Facility of the Center, and by K12 HD01249 to M.D.P. C.P.L. is supported by a postdoctoral fellowship from the German Academic Exchange Service. 
2 Correspondence: Aaron J.W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Room A344, Stanford, CA 94305-5317. FAX: 650 725 7102; aaron.hsueh{at}stanford.edu
3 Current address: Department of Obstetrics and Gynecology, University of Leipzig, Leipzig, Germany.
Accepted: March 5, 2001.
Received: November 8, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  M. Hamel, I. Dufort, C. Robert, C. Gravel, M.-C. Leveille, A. Leader, and M.-A. Sirard Identification of differentially expressed markers in human follicular cells associated with competent oocytes Hum. Reprod., May 1, 2008; 23(5): 1118 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Shkolnik, S. Ben-Dor, D. Galiani, A. Hourvitz, and N. Dekel Molecular characterization and bioinformatics analysis of Ncoa7B, a novel ovulation-associated and reproduction system-specific Ncoa7 isoform Reproduction, March 1, 2008; 135(3): 321 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J Agulleiro, M. Andre, S. Morais, J. Cerda, and P. J Babin High Transcript Level of Fatty Acid-Binding Protein 11 but Not of Very Low-Density Lipoprotein Receptor Is Correlated to Ovarian Follicle Atresia in a Teleost Fish (Solea senegalensis) Biol Reprod, September 1, 2007; 77(3): 504 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jo and T. E. Curry Jr. Luteinizing Hormone-Induced RUNX1 Regulates the Expression of Genes in Granulosa Cells of Rat Periovulatory Follicles Mol. Endocrinol., September 1, 2006; 20(9): 2156 - 2172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Ben-Ami, S. Freimann, L. Armon, A. Dantes, R. Ron-El, and A. Amsterdam Novel function of ovarian growth factors: combined studies by DNA microarray, biochemical and physiological approaches Mol. Hum. Reprod., July 1, 2006; 12(7): 413 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Diouf, K. Sayasith, R. Lefebvre, D. W. Silversides, J. Sirois, and J. G. Lussier Expression of Phospholipase A2 Group IVA (PLA2G4A) Is Upregulated by Human Chorionic Gonadotropin in Bovine Granulosa Cells of Ovulatory Follicles Biol Reprod, June 1, 2006; 74(6): 1096 - 1103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Hourvitz, E Gershon, J D Hennebold, S Elizur, E Maman, C Brendle, E Y Adashi, and N Dekel Ovulation-selective genes: the generation and characterization of an ovulatory-selective cDNA library. J. Endocrinol., March 1, 2006; 188(3): 531 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Dahm-Kahler, C. Lofman, R. Fujii, M. Axelsson, P. O. Janson, and M. Brannstrom An intravital microscopy method permitting continuous long-term observations of ovulation in vivo in the rabbit Hum. Reprod., March 1, 2006; 21(3): 624 - 631. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Perlman, T. Bouquin, B. van den Hazel, T.H. Jensen, H.T. Schambye, S. Knudsen, and J.S. Okkels Transcriptome analysis of FSH and FSH variant stimulation in granulosa cells from IVM patients reveals novel regulated genes Mol. Hum. Reprod., March 1, 2006; 12(3): 135 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. A. Tullet, V. Pocock, J. H. Steel, R. White, S. Milligan, and M. G. Parker Multiple Signaling Defects in the Absence of RIP140 Impair Both Cumulus Expansion and Follicle Rupture Endocrinology, September 1, 2005; 146(9): 4127 - 4137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wang and K. M. J. Menon Regulation of Luteinizing Hormone/Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid Expression in the Rat Ovary: Relationship to Cholesterol Metabolism Endocrinology, January 1, 2005; 146(1): 423 - 431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. McRae, H. M. Johnston, M. Mihm, and P. J. O'Shaughnessy Changes in Mouse Granulosa Cell Gene Expression during Early Luteinization Endocrinology, January 1, 2005; 146(1): 309 - 317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Caetano, R. K. Johnson, J. J. Ford, and D. Pomp Microarray Profiling for Differential Gene Expression in Ovaries and Ovarian Follicles of Pigs Selected for Increased Ovulation Rate Genetics, November 1, 2004; 168(3): 1529 - 1537. [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]
|
|
|
|
|
|
M. D. Pisarska, J. Bae, C. Klein, and A. J. W. Hsueh Forkhead L2 Is Expressed in the Ovary and Represses the Promoter Activity of the Steroidogenic Acute Regulatory Gene Endocrinology, July 1, 2004; 145(7): 3424 - 3433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Hennebold Characterization of the ovarian transcriptome through the use of differential analysis of gene expression methodologies Hum. Reprod. Update, May 1, 2004; 10(3): 227 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sasson, E. Rimon, A. Dantes, T. Cohen, V. Shinder, A. Land-Bracha, and A. Amsterdam Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases Mol. Hum. Reprod., May 1, 2004; 10(5): 299 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. H. Burns, G. E. Owens, S. C. Ogbonna, J. H. Nilson, and M. M. Matzuk Expression Profiling Analyses of Gonadotropin Responses and Tumor Development in the Absence of Inhibins Endocrinology, October 1, 2003; 144(10): 4492 - 4507. [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]
|
|
|
|
|
|
G. Charpigny, M.-J. Leroy, M. Breuiller-Fouche, Z. Tanfin, S. Mhaouty-Kodja, Ph. Robin, D. Leiber, J. Cohen-Tannoudji, D. Cabrol, C. Barberis, et al. A Functional Genomic Study to Identify Differential Gene Expression in the Preterm and Term Human Myometrium Biol Reprod, June 1, 2003; 68(6): 2289 - 2296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. McLean, P. J. Friel, D. Pouchnik, and M. D. Griswold Oligonucleotide Microarray Analysis of Gene Expression in Follicle-Stimulating Hormone-Treated Rat Sertoli Cells Mol. Endocrinol., December 1, 2002; 16(12): 2780 - 2792. [Abstract] [Full Text] [PDF]
|
|
