| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a The Molecular Reproduction Research Laboratory and
b the Experimental Hypertension Group, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
/ß subunits nor the related glycoprotein hormone LH were effective. To determine whether the [Ca2+]i response was due to Ca2+ influx or to intracellular Ca2+ mobilization, cells were exposed to Ca2+-free buffer and to the Ca2+-channel blocker diltiazem (10-5 M). FSH-Induced [Ca2+]i responses were inhibited in Ca2+-free buffer and abrogated in the presence of diltiazem. These novel data demonstrate that FSH can increase [Ca2+]i through L-type voltage-dependent Ca2+ channels via the growth factor type 1 receptor. Our findings support the concept that different receptor motifs act to integrate intracellular signaling events.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Within the gonads, epithelial cells called the Sertoli cell (testis) and granulosa cells (ovary) provide the physical and critical microenvironment required for the development of germinal cells. The function(s) of these cells is directly regulated among other factors by the pituitary glycoprotein hormone, FSH. Other members of this family are pituitary luteinizing hormone (LH), thyroid stimulating hormone (TSH), and placental chorionic gonadotropin (CG) [4,5]. Although these heterodimeric hormones share many structural features and exhibit a common mode of action, they bind to their own unique receptor(s) in respective target cells largely present in the gonads and thyroid gland. However, LH and hCG bind to the same receptor.
The FSH receptor and its counterparts (the LH/CG and TSH receptors) are predicted to share a common structural motif composed of a large N-terminal extracellular domain of about 300 amino acids linked to a region that is similar to the very large family of other G-protein-coupled receptors [6]. The receptor is modeled as having a large extracellular domain linked to a heptahelical structure of membrane spanning segments with consecutive extracellular and intracellular loops. The carboxyl terminal cytoplasmic domain of the receptor consists of about 65–80 amino acids with various sites amenable to phosphorylation. Whereas the extracellular domain has all the information necessary in the form of multiple binding sites to interact with the dimeric configuration of the hormone, the seven-transmembrane domain is thought to help to integrate the receptor into the cell membrane and form a functional unit to transduce the signal along with the cytoplasmic structure. Although there is substantial evidence that cAMP functions as an important second messenger of FSH action in both ovarian granulosa cells [7] and testicular Sertoli cells [8] to activate numerous down-stream signaling events, not all the actions of FSH are completely reproduced by cAMP analogues or other activators of adenylyl cyclase [9,10]. FSH also influences other signal transduction pathways, such as modulation of phosphoinositides and calcium mobilization [11–13].
Intracellular Ca2+ is a critical second messenger that regulates a myriad of cell functions. The mechanisms underlying the induction of calcium signal by FSH in gonadal cells are controversial [10,12,14–17]. Some studies reported that PKA-dependent pathways mediate FSH-induced Ca2+ response [14–17] while others failed to demonstrate a role of cAMP [12]. It is also unclear whether the [Ca2+]i transient is due to mobilization from intracellular sources and/or to Ca2+ influx. The discrepancies among various reports could be attributable in part to the use of primary cultures of reproductive tissues that may contain a heterogeneous collection of cells possessing different receptor molecules of varying structural motifs.
Our current understanding of gonadotropin (glycoprotein hormone) action is based on the concept of a single receptor for each hormone [6]. The full-length FSH receptor, comprising 10 exons and cloned from many species, is known to activate adenylate cyclase [18–20] but not stimulate phosphoinositide [21] or evoke calcium response upon transfection [22,23], suggesting involvement of some other unidentified hormone receptor motif(s) in mediating these well-known effects of FSH in gonadal cells. More recent evidence suggests that other receptor motifs, created by alternative splicing of a single large gene portraying many exons, might also play important role(s) in hormone action. Alternatively spliced receptor transcripts coding for proteins that do not have the heptahelical structure have been reported for all three glycoprotein hormones from different species [23–30], but detailed investigation of their function(s) are limited. Our recent cloning and identification of the FSH receptor protein bearing the features of a growth factor type I receptor in the developing ovary and mature testis [31] prompted the current study, in which we show that this novel receptor motif is highly effective in increasing [Ca2+]i. While partly clarifying this signaling function of FSH, our investigations will serve as a catalyst to recreate functional gonadal cell lines by gene transfection experiments with unique FSH receptor motifs. In this manner, it may be possible to dissect and eventually reconstruct an integrated scheme for hormone signal transduction involving cross talk among various pathways.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All chemicals were of the highest reagent grade available. Fura-2-acetoxymethyl ester and pluronic F-127 were obtained from Molecular Probes Inc. (Eugene, OR). Dimethyl sulfoxide was from Anachemia Canada Inc. (Montreal, PQ, Canada). Dulbecco's modified Eagle's medium, OPTI-MEM, and fetal bovine serum were from Gibco Canada (Mississauga, ON, Canada), and Ham's F-12 medium was from Flow Laboratories Inc. (McLean, VA). The following highly purified hormones used in the tests were prepared in our laboratory according to published procedures. These were ovine FSH (100 x NIH-FSH-S1), deglycosylated FSH (DG-FSH), which acts as a competitive antagonist of the hormone [32], the isolated alpha and beta subunits of FSH, and ovine LH (oLH) (2–3 x NIH-LH-S1).
Cell Culture
The three cDNAs coding for three different types of FSH receptor were reconstructed for expression studies as described recently [29,30], transfected into HEK 293 cells (ATCC CRL 1573), and selected in the presence of G418 (Gibco). For the current investigations we used cells expressing approximately equal numbers of receptors (about 10 000–12 000 per cell) as measured by cell surface binding to intact cells [30,31]. For the [Ca2+]i experiments, using fluorescence digital imaging, the respective cell lines were cultured on glass cover slips (25-mm diameter) in 6-well plates for 2 days in OPTI-MEM medium supplemented with 7.5% fetal bovine serum, at which time they were judged to be about 75% confluent. The medium was removed and washed with incomplete medium (i.e., no serum) and then cultured for 16–24 h to render them quiescent by serum deprivation.
Measurement of Intracellular-Free Ca2+ Concentration
The cells were washed three times with 2 ml of modified Hanks' buffered saline solution and loaded with fura-2 AM (4 µM), which was dissolved in dimethyl sulfoxide with 0.02% pluronic acid [33]. The final dimethyl sulfoxide concentration was less than 0.1% and had no effect on basal [Ca2+]i levels as verified by incubating the untransfected and transfected cells with the vehicle. The cells were incubated for 30 min at 37°C in a humidified incubator (95% air, 5% CO2). After 15 min of incubation to ensure complete de-esterification, cells were finally washed once more with fresh buffer. Under these loading conditions, the ratiometric (343/380 nm) fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentation of fura-2. The coverslip containing cells was placed in a stainless steel chamber and mounted on the stage of an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany). Four glass rings (4- to 5-mm diameter) were placed on the coverslip according to previously described methods [34]. This manipulation allowed for four separate experiments for each coverslip.
Intracellular free Ca2+ concentration was measured in multiple cells simultaneously by fluorescence digital imaging using the Axiovert 135 inverted microscope and Attofluor Digital Fluorescence system (Attofluor Ratiovision, Zeiss). An emission wavelength of 520 nm and alternating excitatory wavelengths of 343 nm and 380 nm were used to measure fura-2 fluorescence. The Attofluor system was calibrated according to previously described methods [34] using the following equation: [Ca2+]i (in mM) = Kd [(R - Rmin)/(Rmax - R)] x ß, where R is the ratio of fluorescence at 343 and 380 nm, Rmax and Rmin are the ratios for fura free acid at 343 and 380 nm in the presence of saturating calcium and zero calcium, respectively, and ß is the ratio of fluorescence of fura-2 at 380 nm in zero and saturating calcium. Kd is the dissociation constant of fura 2 for Ca2+, assumed to be 224 nM. Video images of fluorescence at 520-nm emission were obtained using an intensified charge-coupled device (CCD) Zeiss camera system.
Protocols for Reagent Applications
In initial experiments we compared the effects of FSH on [Ca2+]i in fura-2 loaded cells to select the appropriate cell line for more detailed experiments. As indicated in Figure 1, stable transfected HEK 293 cells expressing the R3 receptor (hereafter referred to as R3 cells) produced the highest and most significant response to 1 nM of FSH. In view of the novel structural features of this receptor, all other studies were performed on this cell line. To determine the source of FSH-stimulated [Ca2+]i responses, experiments were performed in the absence of extracellular Ca2+ and in the presence of voltage-dependent Ca2+ channel blockers. Ca2+-free medium was prepared by the addition of 3 mM EGTA to Hanks' buffer, containing nominal Ca2+ (0.2 mM Ca2+). In a separate series of experiments, diltiazem was used at a concentration of 10-5 M, to block L-type voltage-dependent Ca2+ channels. In these experiments, cells were pre-exposed to Ca2+-free buffer or diltiazem for 15 min prior to addition of hormone.
|
Statistical Analysis
Mean values were calculated for multiple cells in each experiment and then the mean of the experiments was determined and used for analysis. Fifteen to 20 cells were examined in each experimental field, and experiments were repeated 4–8 times. Values are expressed as means ± SEM. Comparison of mean values was performed by Student's t-test where appropriate or by analysis of variance followed by Tukey Kramer's correction for multiple testing. Value of P < 0.05 was considered statistically significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Among the four transcripts identified in our laboratory for the FSH receptor in the ovine testis, three are capable of being expressed on the cell surface to permit verification of their signaling properties. As these have been previously documented in adequate detail [20,29–31], only their salient structural features are depicted in Figure 1 (top panel). The first two receptors termed FSH-R1 and FSH-R2 differ from each other only in the carboxyl terminus, the R2 being shorter by 25 amino acids [29]. FSH-R1 in response to FSH activates adenylate cyclase causing accumulation of cAMP in transfected cells [20], whereas the R2, which also has the seven transmembrane region, inhibits cyclic AMP formation [29]. Since the FSH-R3, unlike R1 and R2, lacks the seven transmembrane region thought to be involved in coupling of such receptors to the effector-G protein assembly for activation [6,18], we presume that a different mechanism may be recruited for signaling. In absence of the heptahelical segment, the R3 receptor utilizes a single putative transmembrane segment like receptors of many growth factor ligands. This receptor expressed on the cell surface binds FSH in a specific manner and with the same affinity as R1 [30,31]. As shown elsewhere, the receptor protein is present in both sheep testis and ovary [31].
Effects of FSH on [Ca2+]i
To assess the receptor type through which FSH may mediate actions on [Ca2+]i, we first compared the response of HEK 293 cells expressing the three different receptors. It became readily apparent that in cells selected to express equivalent number of receptors, which are roughly in the physiological range, the highest response was observed with the cells expressing the FSH-R3 receptor. At 1 nM FSH (~30 ng/ml), cells bearing FSH-R3 produced 2.8-fold increase as compared to a much weaker response of 1.3-fold by those expressing FSH-R1 or no increase at all in the FSH-R2 expressing cells (Fig. 1, lower panel). In untransfected HEK 293 cells or those containing vector cDNA alone, FSH at any concentration had no effect on calcium response (data not shown). Therefore, nonspecific effects may be excluded. In light of these observations we have focused attention on characterizing the response of FSH-R3 expressing cells in more detail.
Rapid Effects and Hormonal Specificity
In R3 cells, FSH induced a rise in [Ca2+]i, with the peak response obtained within 30–60 sec after hormone application (Fig. 2). The [Ca2+]i response was sustained and remained significantly elevated for up to 10 min after stimulation (Fig. 2a). Unlike FSH, neither DG-FSH (tracing b), oLH (tracing c), nor the individual alpha and beta subunits (tracing d) of the hormone had any effect on [Ca2+]i in these cells. At the bottom of Figure 2 are pseudocolor images of the cells responding to 250 ng/ml FSH, where the blue color represents low [Ca2+]i and the red or yellow color represents high [Ca2+]i. Green indicates overlap of blue and red/yellow. These results demonstrate the strict hormonal specificity of the R3 receptor for its effects on [Ca2+]i increase in these cells. They also show that the dissociated individual subunits of the hormone are unable to interact with this receptor and suggest that, as for receptor binding [30], the correct quaternary structure of the hormone is also essential for signaling.
|
FSH Increased [Ca2+]i in a Concentration-Dependent Manner
At a concentration of 60 ng/ml, FSH increased [Ca2+]i to 240 ± 21 nM, and at 1 µg/ml, the hormone increased [Ca2+]i to 927 ± 148 nM (Fig. 3). These responses are in agreement with the high affinity binding of FSH to the R3 receptor as previously reported [30]. Deglycosylated FSH, which is biologically inactive but binds to the receptor as well as does the native glycosylated hormone [32], was unable to stimulate calcium increase at any of the concentrations tested. The rate of response of R3 cells to FSH was ~80% for all concentrations of the hormone tested, indicating that the dose-dependent [Ca2+]i action of FSH is independent of the number of responding cells (Fig. 4). These data obtained with transfected cells are in marked contrast to previous studies with primary cultures of testicular Sertoli cells [17], where increasing concentrations of FSH were reported to recruit a higher percentage of cells to respond by rising [Ca2+]i. A fundamental difference between these two studies is the fact the uniformity of response in our experiments reflect that our cultures contain a single cloned receptor, whereas primary cultures may contain a heterogeneous population expressing a variety of FSH receptors with different signaling characteristics.
|
|
Nature of the [Ca2+]i Response Mediated by R3: (Fig. 5)
In our study, FSH induced a biphasic calcium response, with an initial acute action within 60 sec of hormone stimulation. Recovery to baseline was delayed and 10 min after FSH stimulation [Ca2+]i was still significantly elevated (P < 0.01) above baseline values (137.4 ± 19.9 vs. 68.8 ± 19.9 mmol, stimulated vs. basal). These results obtained with a cloned R3 receptor in passaged cells are different from primary cultures of immature Sertoli cells [17]. The contrasting data in kinetics may be related to differences in cell types and receptor heterogeneity in the latter.
|
Effects of Ca2+-Free Medium and Ca2+ Channel Blockade on FSH-Stimulated [Ca2+]i (Fig. 6)
To determine whether FSH-stimulated [Ca2+]i is mediated via intracellular mobilization and/or via transplasmalemmal Ca2+ influx, we conducted two sets of studies. In the first series (Fig. 6A), experiments were performed to prevent Ca2+ influx into the cells by removing extracellular Ca2+ using the chelating agent EGTA. With different batches of cells, Ca2+ entry was also blocked with the selective L-type voltage-dependent Ca2+ channel blocker, diltiazem, in the presence of Ca2+ (1.2 mM). R3 Cells exposed to Ca2+-free medium failed to respond to FSH, even at the high concentrations of up to 500 ng/ml tested in this series (Fig. 6B). This is in comparison to data shown in Fig. 1, where a low concentration of FSH was shown to produce a robust increase in response in the regular medium containing external calcium ions. The absolute requirement of this became further evident when the Ca2+-free medium in the bath was then replaced with Ca2+-containing medium, and in this instance cells were fully responsive to FSH in the normal manner including sustained elevation for up to 10 min (data not shown). These data show that the agents tested had no deleterious effects on cells. The FSH-induced calcium response of single ovarian granulosa cells derived from pigs is also dependent on external source of calcium in the medium [10] indicating that a receptor system similar to R3 could be serving this function.
|
Diltiazem was selected as the Ca2+ channel blocker of choice in these studies, as it exhibits minimal autofluores-cence, compared to other agents such as verapamil and nifedipine. In cells pretreated with diltiazem in the presence of extracellular Ca2+, FSH failed to induce any change in [Ca2+]i (Fig. 6B). These results indicate that in R3 cells, FSH-stimulated [Ca2+]i responses are mediated via Ca2+ influx, primarily through voltage-dependent Ca2+ channels.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The present investigation using transfected mammalian cells comparing three different types of cloned FSH receptors and studying [Ca2+]i responses in single cells by means of sensitive fluorescence digital imaging techniques, identifies for the first time that a growth factor type I receptor is responsible for mediating this phase of hormone action. FSH has pleiotropic actions on gonadal cells, causing rapid cellular proliferation, inhibition of apoptosis, differentiation, and steroidogenesis [7,36]. The culmination of these events is to sustain optimal production of viable sperm or development of selected (a dominant one in many species including primates) follicle(s) for ovulation. The absolute dependence of these hormone-sustaining actions on the expression of functional FSH receptor(s) is emphasized by our recent targeted disruption experiments on the FSH receptor gene [37] that either causes sterility or reduced fertility.
A question fundamental to the understanding of the mechanism(s) of FSH action is to unravel the nature of the receptor(s) that participate in the signaling pathways. Considering its multiple effects, the issue of a single receptor accounting for all hormonal actions could be subject to challenge. The cloning of a typical seven transmembrane receptor for FSH [18] and other glycoprotein hormones LH and TSH and linkage to the well-known activation of adenylyl cyclase via the Gs system led to the acceptance of the single receptor proposition [6]. Although cAMP is documented to be the main second messenger of the actions of FSH in both the ovary [7] and testis [8] other important signaling events such as [Ca2+]i have also been implicated. The physiological significance of this phenomenon is not completely understood, and there is much controversy regarding the involvement of PKA or other pathways in this process [10,12,14–17].
Based on many studies from our laboratory [27,29–31,38] and others (reviewed in [36]), we suspected that other receptor motifs, created by alternative splicing of the large FSH receptor gene, could mediate some of the known actions of FSH. Differential splicing of DNA in different cell types and stages of growth [2] is a well-established mechanism for generating varying structural protein motifs encrypted in the gene. This mechanism will ensure molecular diversity for cellular regulation in instances where there is only one gene, as in the glycoprotein hormone receptor family. Evidence presented in this study suggests the R3 receptor as a candidate for mediating hormonal effects via the calcium pathway. Among the three cloned receptors that were tested (Fig. 1), the R3 most actively induced a [Ca2+]i response. All the conditions for hormonal specificity and sensitivity were met as shown by the data in Figures 2–5. Our observations on the very weak response (Fig. 1) of the R1 type receptor, which is an efficient stimulator of cAMP in transfected cells [18,20], is in accord with another report [22], which concluded that this receptor does not mediate the influx of calcium into transfected HEK 293 cells. Experimenting with HEK 293 cells that overexpress the R1 type rat receptor to a density 30–40 times that is found normal testicular cells, Shibata et al. [23] found that FSH could not induce inward calcium currents that could be measured by sensitive patch clamp techniques. As the hormone is able to induce this effect in primary cultures of Sertoli or granulosa cells but not in transfected cells expressing R1 on the surface of the cell, these results could suggest that the target cells in the testis and ovary may have a different kind of FSH receptor required to mediate calcium signaling. Our recent cloning and immunological identification of the FSH receptor protein of the growth factor type I receptor in the developing ovary [31] supports the role of an alternative receptor in the mediation of these phenomena. Previous studies suggested that the FSH receptor itself acts as a calcium channel [12] but at that time the existence of receptors of the type R3 in either the testis or ovary was unknown.
In a situation similar to FSH action, calcium rise (signaling) induced by the structurally analogous glycoprotein hormones LH and TSH in their respective target cells or in cells expressing the cloned Gs-coupled receptor is also controversial [39–41]. The demonstration in transfection experiments, that LH receptor signaling by different pathways is dependent on receptor density [41], should add a note of caution in the interpretation of data derived from the use of transfected cells overexpressing excessively large number of receptors. Observations of Shibata et al. [23], who failed to record changes in calcium currents in cells with abundant overexpression of R1 type FSH receptor, clearly suggest involvement of other structures in evoking this response.
The identification of a growth factor type I receptor [31] for a hormone such as FSH, which induces rapid cellular proliferation in the ovary [7] by controlling many cell cycle genes [42], is consistent with the recent discovery of growth factor motifs in gonadotropins. Modeling studies based on x-ray crystallographic data for human chorionic gonadotropin have suggested that the glycoprotein hormones [43] also contain cystine knot motifs that are usually found in growth factors such as NGFs and TGFs. Our previous report showing FSH stimulation of DNA synthesis in a biphasic manner [30] via the R3 receptor in transfected cells lends credence to the existence of a novel receptor-mediated phenomenon. The presence in human thyroid gland of a truncated TSH receptor [28] of a size similar to FSH-R3 and an equivalent carboxyl extension following exon 8 suggests that alternative signaling receptor entities for TSH is a distinct possibility that should be considered.
Other growth promoting hormones such as prolactin and growth hormone, which belong to the cytokine family of regulatory molecules utilizing a single transmembrane type of receptor(s) for signaling, also are known to evoke rapid increases in [Ca2+]i in native and transfected cells [44–46]. These observations and our current data suggest that [Ca2+]i increase mediated by the growth factor type I receptors could be a major mechanism of signaling by hormones that control growth processes. Such actions are an intricate part of normal cellular physiology and neoplastic transformation, and it is known that progression through cell cycle is Ca2+-dependent in many tissues that undergo rapid cell proliferation [47]. These are likely to be very important for growth factor (hormone)-induced increase in transcription, a category to which glycoprotein hormones should also be added. In this regard it should be of interest to reconstruct functional gonadal cell lines by transfecting different FSH receptor types into immortalized ovarian [48] and testicular [49,50] cell lines that have recently become available. These responsive cells should be amenable to investigate and integrate various signaling mechanisms as well as phenotypes and differentiated properties characteristic of gonadal cells.
In conclusion, the present study demonstrates that FSH increases [Ca2+]i by stimulating Ca2+ influx through voltage-dependent Ca2+ channels. These effects are mediated via the growth factor type I receptor that is expressed in the testis and ovary. Our data provide evidence for FSH-induced Ca2+-dependent signaling pathways that may be critical in mediating the cellular effects of the hormone.
|
FOOTNOTES |
|---|
1 This investigation was supported by grants from the Medical Research Council of Canada. R.M.T. is a scholar of the Fonds de la Recherche en Santé du Québec. 
2 Correspondence: M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, PQ, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca
Accepted: November 29, 1999.
Received: September 9, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J.-H. Choi, C.-L. Chen, S. L. Poon, H.-S. Wang, and P. C K Leung Gonadotropin-stimulated epidermal growth factor receptor expression in human ovarian surface epithelial cells: involvement of cyclic AMP-dependent exchange protein activated by cAMP pathway Endocr. Relat. Cancer, March 1, 2009; 16(1): 179 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sen, A. Bettegowda, F. Jimenez-Krassel, J. J. Ireland, and G. W. Smith Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells Endocrinology, September 1, 2007; 148(9): 4400 - 4410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-F. Lin, M.-J. Tseng, H.-L. Hsu, Y.-W. Wu, Y.-H. Lee, and Y.-H. Tsai A Novel Follicle-Stimulating Hormone-Induced G{alpha}h/Phospholipase C-{delta}1 Signaling Pathway Mediating Rat Sertoli Cell Ca2+-Influx Mol. Endocrinol., October 1, 2006; 20(10): 2514 - 2527. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Shukeir, A. Arakelian, G. Chen, S. Garde, M. Ruiz, C. Panchal, and S. A. Rabbani A Synthetic 15-mer Peptide (PCK3145) Derived from Prostate Secretory Protein Can Reduce Tumor Growth, Experimental Skeletal Metastases, and Malignancy-Associated Hypercalcemia Cancer Res., August 1, 2004; 64(15): 5370 - 5377. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Shukeir, A. Arakelian, S. Kadhim, S. Garde, and S. A. Rabbani Prostate Secretory Protein PSP-94 Decreases Tumor Growth and Hypercalcemia of Malignancy in a Syngenic in Vivo Model of Prostate Cancer Cancer Res., May 1, 2003; 63(9): 2072 - 2078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Conti Specificity of the Cyclic Adenosine 3',5'-Monophosphate Signal in Granulosa Cell Function Biol Reprod, December 1, 2002; 67(6): 1653 - 1661. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Manikkam, Y. Li, B. M. Mitchell, D. E. Mason, and L. C. Freeman Potassium Channel Antagonists Influence Porcine Granulosa Cell Proliferation, Differentiation, and Apoptosis Biol Reprod, July 1, 2002; 67(1): 88 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. West, N. E. Carlson, J. S. Lee, A. S. McNeilly, T. P. Sharma, W. Ye, and V. Padmanabhan Acidic Mix of FSH Isoforms Are Better Facilitators of Ovarian Follicular Maturation and E2 Production than the Less Acidic Endocrinology, January 1, 2002; 143(1): 107 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xing and M. R. Sairam Role of CACC-Box in the Regulation of Ovine Follicle-Stimulating Hormone Receptor Expression Biol Reprod, October 1, 2001; 65(4): 1142 - 1149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xing and M. Ram Sairam Characterization of Regulatory Elements of Ovine Follicle-Stimulating Hormone (FSH) Receptor Gene: The Role of E-Box in the Regulation of Ovine FSH Receptor Expression Biol Reprod, February 1, 2001; 64(2): 579 - 589. [Abstract] [Full Text]
|
|
|
|
|
|
P. S. Babu, N. Danilovich, and M. R. Sairam Hormone-Induced Receptor Gene Splicing: Enhanced Expression of the Growth Factor Type I Follicle-Stimulating Hormone Receptor Motif in the Developing Mouse Ovary as a New Paradigm in Growth Regulation Endocrinology, January 1, 2001; 142(1): 381 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Babu, H. Krishnamurthy, P. J. Chedrese, and M. R. Sairam Activation of Extracellular-regulated Kinase Pathways in Ovarian Granulosa Cells by the Novel Growth Factor Type 1 Follicle-stimulating Hormone Receptor. ROLE IN HORMONE SIGNALING AND CELL PROLIFERATION J. Biol. Chem., September 1, 2000; 275(36): 27615 - 27626. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
