help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0332
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sen, A.
Right arrow Articles by Smith, G. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sen, A.
Right arrow Articles by Smith, G. W.
Endocrinology Vol. 148, No. 9 4400-4410
Copyright © 2007 by The Endocrine Society

Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells

Aritro Sen, Anilkumar Bettegowda, Fermin Jimenez-Krassel, James J. Ireland and George W. Smith

Laboratory of Mammalian Reproductive Biology and Genomics (A.S., A.B., F.J.-K., G.W.S.) and Molecular Reproductive Endocrinology Laboratory (F.J.-K., J.J.I.) and Department of Animal Science (A.S., A.B., F.J.-K., J.J.I., G.W.S.), Michigan State University, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: Dr. George W. Smith, Department of Animal Science, Michigan State University, 1230 Anthony Hall, East Lansing, Michigan 48824. E-mail: smithge7{at}msu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We previously demonstrated a negative association of expression of cocaine-and amphetamine-regulated transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling, and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation, and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation, and CART significantly inhibits FSH-induced calcium influx. Select G protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a Go/i-dependent pathway, whereas none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANTRAL FOLLICLE DEVELOPMENT occurs in a wave like pattern in cattle and humans (1, 2, 3, 4). A transient increase in serum FSH precedes the onset of a follicular wave and stimulates emergence of a cohort of small antral follicles. Out of this cohort, typically, a single dominant follicle continues to grow to ovulatory size (5) in the face of declining FSH concentrations and produces markedly greater amounts of estradiol (E). The remaining smaller subordinate follicles lose their capacity to produce E and die via atresia (2, 6, 7, 8). E-producing capacity is lost in ovarian follicles before apoptosis and morphological signs of atresia (7, 9). FSH stimulates multiple granulosa cell (GC) signaling pathways (10, 11, 12), resulting in enhanced E production, cell proliferation, and survival (10, 11, 13, 14). Production of E is critical for follicular growth and triggers the preovulatory gonadotropin surge to promote resumption of meiosis and ovulation (4, 15, 16, 17, 18). Thus, understanding regulation of FSH signaling and intrafollicular E production is critical to enhance knowledge of follicular development and its control.

Whereas the key role of pituitary gonadotropins such as FSH in regulation of ovarian folliculogenesis is well established, the local regulatory molecules that influence gonadotropin action and their cognate signaling pathways are not completely understood. Recent evidence supports a novel local role for a peptide named cocaine- and amphetamine-regulated transcript (CART) in negative regulation of basal E production by bovine GCs and a negative association of CART expression with follicle health status (19). CART mRNA and peptide are expressed by bovine GCs and oocytes, and CART mRNA and follicular fluid concentrations of CART peptide are higher in estrogen-inactive atretic vs. estrogen-active healthy dominant follicles (19). Numerous pleiotropic actions of the CART peptide, including anorexigenic (20, 21, 22, 23), neuroendocrine (24, 25, 26, 27, 28), and antipsychostimulant effects (29, 30, 31), have been described in the brain. To date, the gastrointestinal tract (32), pancreatic cells (33, 34, 35), and ovarian GCs (19) are the only nonneural sites of CART action described. Although identity of the putative CART receptor has not been determined to date, specific saturable binding of CART to AtT20 cells has been described (36). However, the intracellular mechanisms that mediate downstream physiological actions of CART described above are poorly understood.

In this study, we investigated the effects of CART on FSH action in bovine GCs (cAMP accumulation, Ca2+ influx, E production, and aromatase mRNA) and the signaling pathways obligatory to CART action. Results demonstrate CART inhibition of FSH-induced cAMP accumulation, Ca2+ influx, E production, and aromatase mRNA and support involvement of a G protein (Go/i)-dependent pathway in the mechanism of CART action on GCs. Results establish CART as a potent negative regulator of FSH signaling in bovine GCs and directly link CART mechanism of action to regulation of E production, a physiological measure of follicular development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ham’s F12 and MEM-{alpha} media, penicillin and streptomycin, Fungizone-amphotericin B, nonessential amino acids, trypan blue, Superscript II reverse transcriptase, pCR2.1 Topo vector, and Taq DNA polymerase were purchased from Invitrogen Corp. (Carlsbad, CA); phosphatidylinositol 3-kinase (PI3K) inhibitor-LY294002, Akt inhibitor, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA-AM), nifedipine, ionophore A23187, fura-2AM, the Gs{alpha} inhibitor NF449, the Go/i inhibitor NF023, the Gq inhibitor G protein (GP) antagonist-2A, and the MEK1/2 inhibitor U0126 were from Calbiochem (San Diego, CA); active rat CART peptide (55–102) and inactive rat CART peptide (55–76) were from American Peptide Co. (Sunnyvale, CA); forskolin, BSA, apo-transferrin, dithiothreitol (DTT), 3-isobutyl-1-methyl-xanthine, 19 OH-androstenedione, bovine insulin, long R3-IGF-I, EGTA, sodium selenite, and androstenedione were from Sigma Chemical Co. (St. Louis, MO); NIDDK-oFSH-20 was from Harbor-UCLA Research and Education Institute (Los Angeles, CA); Falcon Primaria 96-well plates were from Becton Dickinson and Co. (Lincoln Park, NJ); Catch Point cAMP fluorescent assay kit and Max Gemini EM plate reader with Softmax Pro software were from Molecular Devices (Sunnyvale, CA); RIA kits were from Diagnostic Product Corp. (Los Angeles, CA); RNAqueous microkit and nuclease-free water were from Ambion (Austin, TX); SYBR Green PCR master mix was from Applied Biosciences (Foster City, CA).

Long-term bovine GC culture
Serum-free long-term GC culture was performed as described previously (37), with slight alterations. Briefly, ovaries were obtained at a local abattoir and GCs from 3- to 5-mm follicles collected at random stages of the estrous cycle were pooled together in a 15-ml centrifuge tube containing MEM-{alpha} culture media supplemented with sodium bicarbonate (10 mM), HEPES (20 mM), antibiotics (100 IU/ml penicillin and 0.1 mg/ml streptomycin), Fungizone-amphotericin B (250 µg/ml), nonessential amino acids (1.1 mM), bovine insulin (10 ng/ml), long R3-IGF-I (1 ng/ml), sodium selenite (4 ng/ml), apo-transferrin (5 µg/ml), and androstenedione (10–7 M). The cells were washed with culture media three times, resuspended in 2 ml media, and cell number and viability estimated via trypan blue exclusion.

Cells (1 x 105 viable cells/well) were cultured at 37 C in a humidified atmosphere (5% CO2 and 95% air) for 6 d, with 75% medium replaced every 48 h with fresh medium containing FSH (25 ng/ml unless otherwise mentioned). For experiments involving different signaling inhibitors, GCs were preincubated with 1 or 10 µM dose of inhibitors (described in experiment IV) for 24 h (beginning on d 5 of culture), whereas in experiments with forskolin and 8-bromo-cAMP (8-Br-cAMP), GCs were preequilibrated with media alone (no FSH) for 24 h (d 5) before CART, forskolin, and (or) 8-Br-cAMP treatment. On the sixth day of culture, GCs were treated with media alone or media containing FSH and/or additional treatments (where indicated; various signaling inhibitors/forskolin/8-Br-cAMP/BAPTA-AM/EGTA) in the presence or absence of CART (0.1 µM unless otherwise mentioned) and incubated for 24 h. Media were collected and stored at –20 C for subsequent measurement of E by RIA (19, 38). The cells were washed two times with Dulbecco’s PBS, trypsinized, and cell number determined using a Coulter Counter Particle Z1 (38) (Beckman Coulter, Inc., Fullerton, CA) and viability by trypan blue exclusion. At the end of culture, 75–80% of cells were viable irrespective of treatments (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). After collection, cells were stored at –80 C for measurement of intracellular cAMP (next day) or total RNA isolation. For experiments in which cAMP and E measurements were performed, each treatment was administered in triplicate and cAMP and E levels were normalized to 30,000 cells. For measurements of aromatase mRNA, each treatment was administered to 8 wells per plate, and total RNA was isolated from cells pooled from 4 wells per treatment, resulting in duplicate samples per plate for analysis. All experiments were replicated at least three times.

Measurement of intracellular cAMP
Intracellular cAMP in GCs harvested above (d 7 of culture) was measured using the Catch Point cAMP fluorescent assay kit and Max Gemini EM plate reader with Softmax Pro software according to the manufacturer’s instructions. Increasing amounts of GC lysates (10, 20, and 40 µl) produced a displacement curve parallel to the standard curve. The assay sensitivity was 0.2 nM and all samples were assayed in duplicate.

RIA for E
E concentrations in media were measured using commercially available RIA kits as previously described (38, 39). The assay sensitivity was 0.5 pg/ml and inter- and intraassay coefficients of variation were 7.1 and 7.5%, respectively.

RNA isolation, reverse transcription (RT), and quantitative real-time RT-PCR
RNA isolation, RT, and quantitative real-time RT-PCR analysis were performed as described previously (40, 41) using published primer sequences for aromatase and ß-actin (41, 42) and absolute quantification using standard curve technology. Standard curves for each gene were constructed using 10-fold serial dilutions of corresponding plasmids run on the same plates as samples. For generation of aromatase standard curve, a partial cDNA was amplified from bovine GCs cDNA using the following primers: 5'-CTTATCCTTGCATCCAGACGA-3' forward and 5'-CTAGTAGGCTGCAAATCCATGAG-3' reverse, cloned into the pCR2.1 Topo vector and subjected to fluorescent dye primer sequencing to confirm identity. The plasmid containing ß-actin cDNA was obtained from the Michigan State University Center for Animal Functional Genomics NBFGC cDNA collection and used to construct appropriate standard curves. For each sample, copies of mRNA for aromatase and ß-actin were determined from their respective standard curves and aromatase mRNA abundance normalized relative to abundance of ß-actin mRNA.

Short-term bovine GC culture
Ovaries containing dominant follicles from the first wave of follicular growth were obtained at a local abattoir and GCs from the dominant follicles were isolated and cultured as previously described (39). For each experiment, GCs from each follicle were centrifuged (400 x g for 5 min at 4 C) and washed three times with Ham’s F12 media containing antibiotics (100 IU/ml penicillin and 0.1 mg/ml streptomycin) and 19 OH-androstenedione (1 µM). Cell number and viability were estimated via trypan blue exclusion. Granulosa cells (1 x 105 viable cells/well) were cultured in 96-well plates containing Ham’s F12 medium at 37 C in a humidified atmosphere (5% CO2 and 95% air) for 18 h. After culture, GCs were used for measurement of intracellular calcium concentrations ([Ca2+]i) as described below.

Calcium measurement
To measure [Ca2+]i, GCs were washed twice with standard recording media (S-media) as mentioned previously (43) and loaded with 3 µM fura-2/AM in S-media for 30 min at 37 C. Cells were then washed with S-media and incubated in presence or absence of different treatments (CART/signal inhibitors/DTT/nifedipine; described in experiments III-IV) for an additional 30 min at 37 C before FSH challenge and measurements of [Ca2+]i. Measurement of [Ca2+]i was performed using Max Gemini EM plate reader with Softmax Pro software. Fura-2/AM was excited at 340 and 380 nm and emission measured at 510 nm. The data were digitized at 4-sec intervals for 3 min. Stimulation of GCs with S-media alone or media containing FSH or ionophore (A23187) was begun 60 sec after initiation of recording. All treatments were administered in duplicates, and each experiment was repeated at least three times. Fluorescence ratios at 340/380 nm were calculated and are represented as area under the curve. Any increase in fluorescence ratio that exceeded basal values before FSH stimulation was considered a response.

Statistical analysis
Statistical analyses were performed using a statistical software program from the Statistical Analysis Systems (version 8; SAS Institute, Cary, NC). Data are presented as means ± SEM for all experiments. Generalized linear mixed model analysis using multivariate ANOVA followed by Tukey-Kramer honestly significant difference test was used to determine statistical significance. The data for Ca2+, cAMP, E, and aromatase mRNA were log transformed wherever necessary to meet the assumptions of normality, and for presentation; all means were back-transformed accordingly. A value of P < 0.05 was considered significant.

Experiment I
The long-term GC culture system was used to test the effects of CART on FSH-induced intracellular cAMP and E accumulation and aromatase gene expression. GCs were subjected to the following treatments. Experiment IA included media alone and media containing FSH (5 ng/ml), FSH (25 ng/ml), and FSH (50 ng/ml). For 6 d, 75% medium was replaced every 48 h with fresh medium containing FSH. Experiment IB included media alone and media containing FSH (25 ng/ml); FSH + CART (0.01 µM); FSH + CART (0.1 µM); FSH + CART (1 µM); forskolin (1 µM); forskolin (1 µM) + CART (0.1 µM); 8-Br-cAMP (1 µM); 8-Br-cAMP (1 µM) + CART (0.1 µM); 8-Br-cAMP (10 µM); 8-Br-cAMP (10 µM) + CART (0.1 µM); 8-Br-cAMP (100 µM); and 8-Br-cAMP (100 µM) + CART (0.1 µM). Experiment IC included media alone and media containing FSH (25 ng/ml) and FSH + CART (0.1 µM). All treatments were administered on the sixth day of culture and incubated for 24 h. In experiments involving forskolin and 8-Br-cAMP, GCs were preequilibrated with media alone (no FSH) for 24 h (d 5) before CART, forskolin, and (or) 8-Br-cAMP treatment. Media and cells from experiment IA and IB were used for subsequent measurement of E by RIA and intracellular cAMP, respectively, as described above. Cells from experiment IC were used for RNA isolation, RT, and quantitative real-time RT-PCR.

Experiment II
The calcium requirement for FSH-induced cAMP and E production and inhibitory effects of CART was examined in this study. Granulosa cells from the long-term GC culture system were treated as follows: media alone and media containing BAPTA-AM (1 µM); EGTA (1 mM); FSH (25 ng/ml); FSH (25 ng/ml) + BAPTA-AM (1 µM); FSH (25 ng/ml) + EGTA (1 mM); FSH (25 ng/ml) + CART (0.1 µM); FSH (25 ng/ml) + CART (0.1 µM) + BAPTA-AM (1 µM); and FSH + CART (0.1 µM) + EGTA (1 mM). All treatments were administered on the sixth day of culture and incubated for 24 h. Media and cells were used for subsequent measurement of E by RIA and intracellular cAMP, respectively, as described above.

Experiment III
The effect of CART on FSH-induced rise in [Ca2+]i was tested using the short-term GC culture system. GCs were preincubated with CART and (or) additional treatments (where indicated) 30 min before FSH stimulation. In experiment IIIA, to select an optimal dose of FSH that induced a maximal rise in [Ca2+]i, GCs were stimulated with media alone and media containing FSH (5 ng/ml); FSH (15 ng/ml); FSH (25 ng/ml); and ionophore, A23187 (1 µM). In experiment IIIB, for studies of CART regulation of FSH-induced rise in [Ca2+]i, GCs were treated as follows: media alone and media containing CART (0.1 µM); FSH (25 ng/ml); FSH (25 ng/ml) + CART (0.01 µM); FSH (25 ng/ml) + CART (0.1 µM); and FSH (25 ng/ml) + CART (1 µM). In experiment IIIC, for studies of specificity of CART regulation of FSH-induced rise in [Ca2+]i, GCs were subjected to the following treatments: media containing FSH (25 ng/ml); FSH (25 ng/ml) + CART (0.1 µM); FSH (25 ng/ml) + CART (55–76, inactive; 0.1 µM); FSH (25 ng/ml) + CART (0.1 µM) + DTT (5 mM); and FSH (25 ng/ml) + DTT (5 mM). GCs were incubated in presence or absence of the above-mentioned treatments for 30 min at 37 C before FSH challenge followed by measurements of [Ca2+]i as described above.

Experiment IV
The putative signaling pathways and mediators of the negative effects of CART on FSH-induced Ca2+ signaling were examined as follows: GCs cultured in the presence or absence of CART (0.1 µM) were subjected to the following treatments: experiment IVA, media containing FSH (25 ng/ml), FSH (25 ng/ml) + Gs{alpha} inhibitor NF449, FSH (25 ng/ml) + Go/i inhibitor NF023, and FSH (25 ng/ml) + Gq inhibitor GP antagonist-2A; experiment IVB, media containing FSH (25 ng/ml), FSH (25 ng/ml) + CART (0.1 µM), FSH (25 ng/ml) + nifedipine (50 µM), and FSH (25 ng/ml) + nifedipine (50 µM) + CART (0.1 µM). GCs were incubated in presence or absence of the above-mentioned treatments for 30 min at 37 C before FSH challenge followed by measurements of [Ca2+]i as described above. To further investigate the involvement of various signaling molecules in the negative effects of CART on FSH-induced cAMP and E production, GCs from long-term culture system were treated as follows: experiment IVC, media alone and media containing FSH (25 ng/ml); FSH (25 ng/ml) + CART (0.1 µM); Go/i inhibitor NF023; FSH + Go/i inhibitor NF023; FSH (25 ng/ml) + CART (0.1 µM) + Go/i inhibitor NF023; MEK1/2 inhibitor U0126; FSH (25 ng/ml) + MEK1/2 inhibitor U0126; FSH (25 ng/ml) + CART (0.1 µM) + MEK1/2 inhibitor U0126; PI3K inhibitor-LY294002; FSH (25 ng/ml) + PI3K inhibitor-LY294002; FSH (25 ng/ml) + CART (0.1 µM) + PI3K inhibitor-LY294002; Akt inhibitor; FSH (25 ng/ml) + Akt inhibitor; and FSH (25 ng/ml) + CART (0.1 µM) + Akt inhibitor. GCs were preincubated with 1 or 10 µM dose of inhibitors for 24 h (beginning on d 5 of culture), and on the sixth day of culture, GCs were treated with media alone or media containing FSH and/or the signaling inhibitors in presence or absence of CART and incubated for 24 h. Media and cells from experiment IVC were then used for subsequent measurement of E by RIA and intracellular cAMP, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of CART on FSH-induced intracellular cAMP and E accumulation and aromatase gene expression
It is well established that FSH activates the cAMP-protein kinase A pathway leading to aromatase gene expression and E production (10, 11, 44, 45). Thus, the aim of described studies was to investigate whether CART treatment regulates above actions of FSH in bovine GCs.

Dose responsiveness of GCs to FSH was established to determine optimal stimulatory dose of FSH for subsequent studies of CART regulation of FSH signaling. In experiment IA, a maximal increase in intracellular cAMP (2-fold, P < 0.01; Fig. 1AGo) and E (20-fold, P < 0.01; Fig. 1BGo) accumulation was observed in response to 25 ng/ml of FSH. Thus, this dose was selected for all subsequent experiments using this culture system.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 1. Responsiveness of bovine GCs to different doses of FSH (0, 5, 25, 50 ng/ml) with respect to intracellular cAMP concentration (A) and E levels (B) in culture media. GCs were treated with different amounts of FSH for 7 d with 75% media replaced with fresh media (supplemented with 10–7 M androstenedione) every 48 h. cAMP and E levels were measured in GCs and media, respectively, as described in Materials and Methods. Data were normalized to 30,000 cells, and bars (mean ± SEM of results of three replicate experiments) with different superscripts denote significant differences across treatments (a vs. b, b vs. c, and a vs. c, P < 0.01).

 
FSH-induced intracellular cAMP (Fig. 2AGo) and E accumulation (Fig. 2BGo) were reduced in experiment 1B in response to 24 h preincubation with increasing concentrations of CART (0, 0.01, 0.1, and 1 µM). A concentration-dependent decrease of 1.7- (P < 0.01) and 4.1 (P < 0.01)-fold in cAMP concentrations (Fig. 2AGo) was observed in response to treatment with 0.01 and 0.1 µM of CART, respectively, whereas 1 µM CART did not further reduce cAMP levels. In contrast, all doses of CART decreased (P < 0.01) the FSH induced increase in E (Fig. 2BGo). CART had no effect on basal cAMP and E accumulation in the absence of FSH.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 2. Effects of different CART concentrations (0, 0.01, 0.1, 1 µM) on the FSH-induced increase in intracellular cAMP concentration (A) and E levels (B) in culture media. GCs were treated with 25 ng/ml of FSH for 6 d and then preincubated with different CART concentrations plus FSH (25 ng/ml). cAMP and (or) E levels were measured in GCs and media collected on d 7 as described in Materials and Methods. Data were normalized to 30,000 cells, and bars (mean ± SEM of results of three replicate experiments) with different superscripts denote significant differences across treatments (a vs. b, b vs. c, and a vs. c, P < 0.01).

 
Furthermore, to investigate whether the negative effects of CART on cAMP and E are specific to FSH treatment, the effects of CART on cAMP and E accumulation in response to pharmacological activation of adenylate cyclase were investigated (experiment IB). Treatment with forskolin (1 µM) mimicked the effects of FSH and stimulated an increase in cAMP and E accumulation over basal levels, whereas pretreatment with 0.1 µM CART inhibited (P < 0.01) the forskolin-induced increase in cAMP and E levels (Fig. 3Go, A and B). To investigate whether inhibitory effects of CART on E production are also potentially manifest downstream of cAMP accumulation, GCs were supplemented with different concentrations of 8-Br-cAMP (0, 1, 10, and 100 µM) in the presence or absence of CART. All the concentrations of 8-Br-cAMP tested mimicked the effects of FSH and stimulated an increase in E accumulation over untreated controls (P < 0.01). Pretreatment (24 h) of GCs with CART significantly (P < 0.01) reduced both FSH and 8-Br-cAMP-induced E accumulation. The inhibitory effects of CART were less pronounced when GCs were treated with 10 and 100 µM 8-Br-cAMP, compared with FSH alone or 1 µM 8-Br-cAMP (P < 0.01).


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 3. Effects of CART on the forskolin-induced increase in intracellular cAMP concentration (A), E levels in culture media (B), and 8-Br-cAMP-induced E accumulation (C) in culture media. GCs were treated with 25 ng/ml of FSH for 5 d and then preequilibrated with media alone (no FSH; control) for 24 h before addition (on d 6) of forskolin (1 µM) or 8-Br-cAMP (1, 10, and 100 µM) with or without CART (0.1 µM). cAMP and E levels were measured in GCs and media collected on d 7 as described in Materials and Methods. Data were normalized to 30,000 cells, and bars (mean ± SEM of results of three replicate experiments) with different superscripts denote significant differences across treatments (A and B: a vs. b and b vs. c, P < 0.01, a vs. c, P < 0.05; C: a vs. b, b vs. c, and a vs. c, P < 0.01).

 
Aromatase is an FSH-regulated, rate-limiting enzyme in GC E production (46). Treatment of GCs with FSH (experiment IC) resulted in a 5-fold increase in aromatase mRNA abundance (P < 0.05). Treatment with CART (0.1 µM) reduced aromatase mRNA abundance in FSH-treated cells (P < 0.05) to basal levels (Fig. 4Go).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 4. Quantitative real-time RT-PCR analysis of effects of CART on FSH-induced aromatase mRNA abundance in bovine GCs. Cells were cultured for 7 d (Materials and Methods) with FSH (0 ng/ml), FSH (25 ng/ml), and FSH (25 ng/ml) + CART (0.1 µM; administered on d 6). Data were normalized relative to abundance of ß-actin mRNA in the same samples, and bars (mean ± SEM of results of three replicate experiments) with different superscripts denote significant differences across treatments (a vs. b, P < 0.05).

 
Results of the above experiments demonstrate for the first time that CART negatively affects FSH-induced cAMP accumulation and relevant downstream physiological responses, especially aromatase mRNA and E production in bovine GCs.

Calcium requirement for FSH-induced cAMP and E production and inhibitory effects of CART
Calcium involvement in regulation of steroidogenesis in other cell types is well established (47, 48) as is FSH regulation of GC Ca2+ influx in other species (49, 50, 51). Furthermore, CART inhibition of K+ and ionophore-induced Ca2+ influx in hippocampal neurons (52) has been reported. Consequently, to determine the potential significance of regulation of [Ca2+]i to CART action, we determined whether Ca2+ is obligatory for FSH-induced cAMP and E production and the potential inhibitory effects of CART (experiment II). Treatment of GCs with the Ca2+ chelators BAPTA-AM (1 µM) and EGTA (1 mM) completely blocked the ability of FSH to increase intracellular cAMP levels (P < 0.01; Fig. 5AGo) and E production (P < 0.01; Fig. 5BGo), thereby demonstrating a requirement of Ca2+ for FSH-induced steroidogenesis in bovine GC. BAPTA-AM did not alter basal cAMP (Fig. 5AGo) and E levels (Fig. 5BGo) in the absence of FSH, whereas EGTA reduced basal cAMP concentrations (Fig. 5AGo; P < 0.01) but had no effect on basal E production (Fig. 5BGo). Furthermore, both Ca2+ chelators masked potential inhibitory effects of CART on FSH-stimulated cAMP and E accumulation in bovine GCs. No effect of BAPTA-AM or EGTA treatment on cell viability was observed (supplemental Table 1). The above experiment clearly demonstrates that Ca2+ is required for FSH-induced cAMP accumulation and E production in bovine GCs, and thus, inhibitory effects of CART on FSH action were masked by Ca2+ chelator treatment.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 5. Effect of Ca2+ chelators on CART inhibition of FSH-induced intracellular cAMP (A) and E levels (B) in culture media. GCs were treated with 25 ng/ml of FSH for 6 d and BAPTA-AM (1 µM) or EGTA (1 mM) was added 24 h (on d 5 of culture) before CART treatment. On d 6 of culture, GCs were treated with media alone or media containing FSH (25 ng /ml) and (or) the Ca2+ chelators in the presence or absence of CART (0.1 µM) and incubated for 24 h. cAMP and E levels were measured in GCs and media collected on d 7 as described in Materials and Methods. Data were normalized to 30,000 cells, and bars (mean ± SEM of results of three replicate experiments) with different superscripts denote significant differences across treatments (a vs. b, b vs. c, and a vs. c, P < 0.01).

 
Effect of CART on FSH-induced rise in [Ca2+]i
We next investigated whether CART can directly inhibit FSH-induced Ca2+ signaling using a short-term GC culture system. To select the optimal dose of FSH (experiment IIIA) to elicit a maximal increase in [Ca2+]i, GCs were stimulated with different doses of FSH (0, 5, 15, and 25 ng/ml). FSH stimulation induced a dose-dependent linear increase in [Ca2+]i (P < 0.05) in GCs (Fig. 6AGo). However, the highest FSH dose (50 ng/ml; data not shown) did not have any further effect. The rise in [Ca2+]i was specific and initiated only in response to FSH treatment, as illustrated by representative traces of the Ca2+ response for cultures stimulated with vehicle media alone or vehicle media containing the lowest dose of FSH tested (5 ng/ml; Fig. 6BGo). A response was also elicited when GCs were stimulated with ionophore A23187 as a positive control (supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Because 25 ng/ml FSH induced a maximal increase in [Ca2+]i, this dose was selected for all subsequent [Ca2+]i experiments. Furthermore, when Ca2+ experiments were performed in Ca2+-free media, stimulation of GCs with FSH (25 ng/ml) failed to elevate [Ca2+]i above prestimulation levels, demonstrating that the FSH-induced rise in [Ca2+]i in bovine GCs is mediated via Ca2+ influx (supplemental Fig. 1B).


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 6. Effect of FSH on [Ca2+]i in bovine GCs from short-term culture system. A, Summary of changes in [Ca2+]i in response to different doses of FSH (0, 5, 15, 25 ng/ml). Values are presented as the mean ± SEM of area under the curve of the Ca2+ responses for three replicate experiments, and different superscripts denote significant differences across treatments (a vs. b, b vs. c, and c vs. d, P < 0.05; a vs. c, a vs. d, and b vs. d, P < 0.01). B, Representative profiles of the Ca2+ responses induced by media alone and media containing the lowest stimulatory dose of FSH tested (5 ng/ml). The arrow represents the time point of FSH stimulation. GCs were isolated and prepared for fura-2 AM measurement of [Ca2+]i as described in Materials and Methods. Data are depicted as the relative fluorescence ratio (340:380 nm) over time (in seconds).

 
The FSH-induced rise in [Ca2+]i was reduced (P < 0.05) in response to 30 min preincubation (experiment IIIB) with 0.1 and 1 µM CART (1.6-fold) but not 0.01 µM CART. The higher dose of CART (1 µM) did not further decrease [Ca2+]i beyond that observed in response to 0.1 µM CART (Fig. 7AGo). CART pretreatment (0.1 µM) did not alter basal Ca2+ levels (Fig. 7AGo). Representative profiles of the Ca2+ responses induced by FSH with or without CART (0.1 µM) are depicted in Fig. 7BGo.


Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 7. Effect of CART on the FSH-induced increase in [Ca2+]i in bovine GCs from short-term culture system. A, Summary of effects of CART on basal and FSH-induced changes in [Ca2+]i. GCs were preincubated with CART (0, 0.01, 0.1, and 1 µM) for 30 min before stimulation with media alone or media containing FSH (25 ng/ml) as described in Materials and Methods. Values are presented as the mean ± SEM of area under the curve of the Ca2+ responses for three replicate experiments, and different superscripts denote significant differences across treatments (a vs. b and a vs. c, P < 0.01; b vs. c, P < 0.05). B, Representative profiles of the Ca2+ responses induced by media containing FSH (25 ng/ml) in the presence or absence of CART peptide (0.1 µM). The arrow represents the time point of FSH stimulation. The cells were isolated and prepared for fura-2 AM measurement of [Ca2+]i as described in Materials and Methods. Data are depicted as the relative fluorescence ratio (340:380 nm) over time (in seconds).

 
To demonstrate the specificity (Fig. 8Go) of CART inhibition of the FSH-induced increase in [Ca2+]i, GCs were preincubated (experiment IIIC) with the biologically active CART peptide (55–102; 0.1 µM), an inactive CART peptide (55–76; 0.1 µM), or with the biologically active CART peptide (55–102) pretreated with DTT to reduce intramolecular disulfide bonds critical to biological activity (36). Only the active CART peptide (55–102) significantly inhibited (1.9-fold) the FSH-induced rise in [Ca2+]i (P < 0.05; Fig. 8Go). Collectively, results demonstrate that CART inhibits hormone (FSH)-induced Ca2+ influx in bovine GCs. Given the demonstrated requirement of Ca2+ for FSH-induced cAMP accumulation and E production, results support a potential role for suppression of Ca2+ influx in mediating the negative effects of CART on FSH signaling in bovine GCs.


Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 8. Specificity of CART inhibition of the FSH-induced rise in [Ca2+]i. GCs were preincubated for 30 min with active CART (55–102) (0.1 µM) or inactive CART (55–76) peptides (0.1 µM) or with active CART (55–102) (0.1 µM) that was preincubated (1 h) with DTT (5 mM) or with DTT (5 mM) alone as described in Materials and Methods and then stimulated with FSH (25 ng/ml). Values are presented as the mean ± SEM of area under the curve of the Ca2+ responses for five replicate experiments, and different superscripts denote significant differences across treatments (a vs. b, P < 0.05).

 
Putative signaling pathways mediating the negative effects of CART on FSH action
To investigate the underlying intracellular mechanisms responsible for CART actions on GCs, the requirement of various G proteins for CART regulation of FSH-induced Ca2+ influx was determined using commercially available pharmacological inhibitors. Pretreatment (30 min) with the Go/i inhibitor completely abolished CART inhibition of the FSH-induced rise in [Ca2+]i (Fig. 9AGo). In contrast, the Gs{alpha} inhibitor, irrespective of CART treatment, significantly (P < 0.05) reduced the FSH-induced rise in [Ca2+]i. No effect of the Gq inhibitor was observed on CART regulation of FSH-induced Ca2+ influx (experiment IVA). As mentioned in Materials and Methods, two concentrations (1 µM, Fig. 9AGo, and 10Go µM, data not shown) of each inhibitor were used and similar effects observed.


Figure 9
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 9. A, Effect of G protein inhibitors on CART inhibition of the FSH-induced rise in [Ca2+]i. GCs were preincubated (30 min) with inhibitors (1 µM) of different G proteins (the Gs{alpha} inhibitor NF449, the Go/i inhibitor NF023, and the Gq inhibitor GP antagonist-2A in the presence or absence of CART (0.1 µM) before stimulation with FSH (25 ng/ml) as described in Materials and Methods. Values are presented as the mean ± SEM of area under the curve of the Ca2+ responses for three replicate experiments, and different superscripts denote significant differences across treatments (a vs. b and b vs. c, P < 0.05; a vs. c, P < 0.01). B, Effect of CART and an L-type Ca2+ channel antagonist on FSH-induced Ca2+ influx. Experiments were performed in media containing Ca2+ and GCs were preincubated (30 min) with or without CART (0.1 µM) in the presence or absence of the L-type Ca2+ channel antagonist nifedipine (50 µM) and then stimulated with FSH (25 ng/ml) as described in Materials and Methods. Values are presented as the mean ± SEM of area under the curve of the Ca2+ responses for four replicate experiments, and different superscripts denote significant differences across treatments (a vs. b and b vs. c, P < 0.05; a vs. c, P < 0.01).

 

Figure 10
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 10. Effect of inhibitors of select signaling proteins (Go/i inhibitor NF023, PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126, and Akt inhibitor) on CART inhibition of the FSH-induced rise in intracellular cAMP concentration (A) and E levels (B) in culture media. GCs were treated with FSH (25 ng/ml) for 6 d and preincubated with above inhibitors (1 µM) for 24 h before CART treatment. On d 6 of culture, GCs were treated with media alone or media containing FSH (25 ng/ml) and (or) signaling inhibitors (1 µM) in presence or absence of CART (0.1 µM) and incubated for 24 h. cAMP and E levels were measured in GCs and media collected on d 7 as described in Materials and Methods. Data were normalized to 30,000 cells, and bars (mean ± SEM for three replicate experiments) with different superscripts denote significant differences across treatments (A, P < 0.01; B, P < 0.01, a, b).

 
FSH stimulation of L-type Ca2+ channel activity has been previously established (49, 50, 51, 53). Thus, we investigated the potential involvement of voltage-gated L-type Ca2+ channels in CART regulation of FSH-induced Ca2+ influx in bovine GC (experiment IVB). Based on a nifedipine (L-type Ca2+ channel antagonist) dose response experiment (0, 10, 50, and 100 µM) in the presence or absence of CART (data not shown), a maximal inhibitory dose of nifedipine (50 µM) was selected. Treatment of GCs with nifedipine (50 µM) in the presence of FSH mimicked the inhibitory effects of CART on FSH-stimulated Ca2+ influx (Fig. 9BGo). Nifedipine reduces the opening frequency and duration of voltage-gated L-type Ca2+ channels (54). Furthermore, preincubation of GCs with nifedipine and CART further decreased Ca2+ levels by 1.6-fold (P ≤ 0.05; Fig. 9BGo). Results indicate that CART inhibition of FSH-induced Ca2+ influx in bovine GCs may be mediated at least in part by inhibition of L-type Ca2+ channels.

Subsequent experiments examined the requirement of various signaling molecules for the negative effects of CART on FSH-induced cAMP and E production (experiment IVC). Pretreatment with the Go/i inhibitor completely abolished the inhibitory effect of CART on FSH-induced cAMP accumulation but did not alter basal or FSH-induced cAMP accumulation in the absence of CART (Fig. 10AGo). No effect of the MEK1/2 inhibitor or the Akt inhibitor on basal and FSH-stimulated cAMP accumulation in the presence or absence of CART was observed (Fig. 10AGo). In contrast to the complete reversal of CART-induced inhibition of FSH-stimulated cAMP accumulation observed in cells pretreated with the Go/i inhibitor, only a partial reversal of the negative effects of CART on FSH-induced E production was observed when Go/i was inhibited (P < 0.05; Fig. 10BGo). Similar results were observed when cells were treated with a higher dose of the inhibitor (10 µM; data not shown). No effect of blocking Go/i on FSH-stimulated E production in the absence of CART was observed (Fig. 10BGo). In contrast, pharmacological inhibition of PI3K, MEK1/2, and Akt completely blocked the FSH-induced increase in E production, demonstrating a requirement of these signaling molecules for FSH-induced E production (Fig. 10BGo) and masked potential inhibitory effects of CART on FSH-induced E production. Thus, results demonstrate that inhibitory effects of CART on FSH-induced Ca2+ influx, cAMP accumulation, and E production are mediated by a Go/i-dependent pathway but also support a potential involvement of Go/i-independent pathways in the mechanism of action of CART.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fertility in humans and other mammals depends on sustained production of E by ovulatory follicles. However, more than 99% of ovarian follicles lose E-producing capacity and die at various stages of folliculogenesis. It is well established that in GCs, FSH activates the cAMP-protein kinase A pathway that in turn induces aromatase gene expression and E synthesis (10, 11, 44, 45). However, little is known about the intrafollicular inhibitory factors and underlying mechanisms negatively regulating E production. This study for the first time demonstrates novel potent inhibitory effects of CART on FSH-induced E production and multiple upstream components of the FSH signaling pathway critical to regulation of steroidogenesis. The most significant findings of this study were the inhibitory effects of CART on FSH-induced cAMP levels, [Ca2+]i, aromatase gene expression, and E production. Furthermore, described effects of CART on FSH signaling were shown to be G protein (Go/i) dependent. Given the importance of E within and outside the reproductive system and the multitude of biological actions of CART in the central nervous system and periphery, results also provide novel information on CART signaling linked to a relevant downstream biological response (E production).

The inhibitory actions of CART on cAMP accumulation are not likely mediated solely at the level of the FSH receptor (heterologous desensitization) because CART also inhibited the increase in cAMP and E induced by pharmacological activation of adenylate cyclase with forskolin treatment. This suggests that the inhibitory effects of CART on FSH-induced cAMP accumulation may involve inhibition of adenylate cyclase and/or stimulation of phosphodiesterase expression/activity in bovine GCs. In contrast to our results, a recent study demonstrated CART enhancement of insulin secretion and cAMP accumulation in INS-1(832/13) cells stimulated with 3-isobutyl-1-methyl-xanthine or glucagon like peptide-1 in the presence of glucose (34). Reasons for the differential effects of CART on cAMP accumulation in GCs vs. ß-cells are unclear. There are precedents for differential regulation of cAMP synthesis by the same factor in different cell types/species (55, 56). Also, differential CART regulation of specific adenylate cyclase and phosphodiesterase isoforms (44, 57) in the two cell types and (or) differential effects of acute (ß-cells) vs. 24 h CART stimulation used in the present studies cannot be discounted. More importantly, the ability of CART to inhibit 8-Br-cAMP-induced E production clearly suggests that the ability of CART to inhibit FSH-induced E production is not mediated solely via inhibition of cAMP accumulation but rather that CART may also regulate other FSH-induced signaling proteins downstream of cAMP that are essential for E production. Previous studies (58, 59, 60, 61, 62) as well as results of the current study (Fig. 10BGo) support the requirement of PI3K, MEK1/2, and Akt activity for FSH-induced E production. However, whether CART regulates the expression or activity of any of these downstream protein kinases is not known and will require further investigation.

Our results demonstrate that the inhibitory effects of CART on FSH-stimulated cAMP accumulation and Ca2+ influx are via a Go/i-dependent mechanism. CART signaling events in the brain and a pituitary cell line (AtT20) have also been shown to be Go/i dependent. CART inhibition of K+-induced Ca2+ influx in hippocampal neurons and stimulation of ERK1/2 activation in AtT20 cells are blocked via pertussis toxin treatment (52, 63). However, the inhibitory effects of CART on FSH-stimulated E production in the present studies were only partially blocked after treatment with the Go/i inhibitor. This suggests that CART inhibition of FSH-induced E production could involve Go/i-dependent and -independent mechanisms. Further insight into potential Go/i-dependent and -independent mechanisms of CART signaling would be best resolved via cloning and characterization of the putative CART receptor on GCs. However, the identity of the CART receptor is still unknown, even though specific binding of CART to AtT20 cells has been demonstrated (36). Our results suggest that CART may act via a receptor linked to Go/i, but involvement of other convergent pathways in the mechanism of action of CART cannot be ruled out.

CART-induced suppression of a trophic hormone (FSH)-stimulated increase in [Ca2+]i has not been reported previously. The complete functional significance of CART suppression of [Ca2+]i to its mechanism of action in inhibition of FSH-stimulated cAMP accumulation and E production remains to be established. However, our results clearly demonstrate that Ca2+ is required for the FSH-induced increase in cAMP levels and E production. Whether CART inhibition of cAMP and E production is due to direct interaction of CART activated Go/i protein with adenylyl cyclase/phosphodiesterase or due to the decrease in [Ca2+]i is not known. There is evidence that both Go/i proteins and Ca2+ regulate the activity/expression of different isoforms of adenylyl cyclase/phosphodiesterase, which in turn regulate cAMP generation/accumulation and steroidogenesis (44, 64).

Evidence from the current studies also supports a potential role for regulation of L-type Ca2+ channels in the mechanism of action of CART. In rat and ovine GCs Ca2+ entry through L-type channels is required for FSH actions (58, 65). Yermolaieva et al. (52) reported that nifedipine treatment blocks CART regulation of K+-induced Ca2+ signaling in hippocampal primary cell cultures. However, in the present study, we also demonstrated that preincubation of GC with nifedipine and CART further decreased [Ca2+]i when compared with treatment with CART or nifedipine alone. This suggests that in addition to L-type Ca2+ channels, CART may also regulate activity of other Ca2+ channels. Expression of L- and T-type Ca2+ channels in GCs has been reported previously (65, 66, 67, 68). There is also evidence of Go/i--dependent inhibition of L-, N-, and P/Q-type Ca2+ channels in rat lateral hypothalamic neurons (69). Thus, further studies are needed to identify other Ca2+ channels in GCs that might be regulated by CART.

It is also important to note that inhibition of Gs{alpha}, irrespective of CART treatment, greatly reduced the FSH-induced rise in [Ca2+]i, suggesting that FSH-induced Ca2+ influx in bovine GC is Gs{alpha} dependent. A recent study (70) reported that FSH-induced Ca2+ influx in rat Sertoli cells is mediated by a G{alpha}h-dependent pathway, and no effect of inhibition of Gs{alpha} on FSH-induced Ca2+ influx was observed. The reasons for the discrepancy between results of the present study and the study in rat Sertoli cells are not known. It is possible that multiple G proteins are linked to the R3 isoform of the FSH receptor, the main isoform involved in FSH-induced Ca2+ influx (50, 51). In the present study, [Ca2+]i in cells treated with the Gs{alpha} inhibitor were significantly lower than in cells cultured in the absence of the inhibitor but higher than basal values and thus still increased in response to FSH treatment. The observed partial inhibitory effect of blocking Gs{alpha} cannot be attributed to dose of inhibitor used because similar results were observed when higher (10 µM) concentrations of the inhibitor were used. Thus, Gs{alpha}-independent mechanisms of FSH-induced Ca2+ influx in bovine GCs cannot be discounted.

Whereas not affecting FSH-induced cAMP accumulation, inhibition of PI3K, MEK1/2, and Akt completely blocked FSH-induced E production. The above-mentioned kinases are known to be important for follicular development and regulate expression of genes necessary for cell survival and steroidogenesis (58, 59, 60, 61, 62). In rat GCs, FSH-stimulated Erk activation is MEK1/2 and Ca2+ dependent (65, 71). Our data demonstrate that activity of such kinases is required for FSH-induced E production independent of cAMP accumulation. Thus, it is not possible from the present studies to interpret whether CART regulates the above-mentioned kinases/pathways. However, studies in the brain and AtT20 cells have demonstrated that acute CART treatment results in Erk activation (63, 72). In these studies, Erk activation was observed within 2–5 min of CART treatment, whereas activated Erk levels dropped within 10 min to basal levels.

In summary, this study for the first time provides direct evidence of CART inhibition (via a Go/i dependent pathway) of multiple components of the FSH signaling cascade in bovine GCs critical to E production. Results illustrate the utility of described model system for studies of CART signaling pathways critical to a relevant downstream biological response and form the foundation for future studies of CART regulation of other FSH-responsive target genes/components of FSH action and the involvement of additional pathways in CART signaling.


   Acknowledgments
 
The authors thank Janet Ireland and Lihua Lv for their help with follicle cutting and Nora Bello for all her help with the statistical analysis.


   Footnotes
 
This work was supported by the Michigan Agricultural Experiment Station, a seed grant from the Michigan State University Reproductive and Developmental Sciences Program, and National Research Initiative Competitive Grant 2005-35203-16011 from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online June 14, 2007

Abbreviations: BAPTA-AM, Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester; 8-Br-cAMP, 8-bromo-cAMP; [Ca2+]i, intracellular calcium concentrations; CART, cocaine- and amphetamine-regulated transcript; DTT, dithiothreitol; E, estradiol; GC, granulosa cell; GP, G protein; PI3K, phosphatidylinositol 3-kinase; RT, reverse transcription.

Received March 9, 2007.

Accepted for publication June 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ginther OJ, Beg MA, Bergfelt DR, Donadeu FX, Kot K 2001 Follicle selection in monovular species. Biol Reprod 65:638–647[Abstract/Free Full Text]
  2. Baerwald AR, Adams GP, Pierson RA 2003 Characterization of ovarian follicular wave dynamics in women. Biol Reprod 69:1023–1031[Abstract/Free Full Text]
  3. Sirois J, Fortune JE 1988 Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol Reprod 39:308–317[Abstract]
  4. Fortune JE 1994 Ovarian follicular growth and development in mammals. Biol Reprod 50:225–232[Abstract]
  5. Fortune JE, Rivera GM, Yang MY 2004 Follicular development: the role of the follicular microenvironment in selection of the dominant follicle. Anim Reprod Sci 82–83:109–126
  6. Mihm M, Good TE, Ireland JL, Ireland JJ, Knight PG, Roche JF 1997 Decline in serum follicle-stimulating hormone concentrations alters key intrafollicular growth factors involved in selection of the dominant follicle in heifers. Biol Reprod 57:1328–1337[Abstract]
  7. Sunderland SJ, Crowe MA, Boland MP, Roche JF, Ireland JJ 1994 Selection, dominance and atresia of follicles during the oestrous cycle of heifers. J Reprod Fertil 101:547–555[Abstract/Free Full Text]
  8. Richards JS, Hedin L 1988 Molecular aspects of hormone action in ovarian follicular development, ovulation, and luteinization. Annu Rev Physiol 50:441–463[CrossRef][Medline]
  9. Austin EJ, Mihm M, Evans AC, Knight PG, Ireland JL, Ireland JJ, Roche JF 2001 Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle. Biol Reprod 64:839–848[Abstract/Free Full Text]
  10. Richards JS 2001 Perspective: the ovarian follicle—a perspective in 2001. Endocrinology 142:2184–2193[Free Full Text]
  11. Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH, Falender AE, Lo YK, Sharma SC 2002 Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Prog Horm Res 57:195–220[Abstract/Free Full Text]
  12. Woods JR, Strauss JF 2002 Multiple signal transduction pathways regulate ovarian steroidogenesis. Rev Endocr Metab Disord 3:33–46[CrossRef][Medline]
  13. McGee EA, Hsueh AJW 2000 Initial and cyclic recruitment of ovarian follicles. Endocr Rev 21:200–214[Abstract/Free Full Text]
  14. Wandji SA Eppig J, Fortune JE 1996 FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles. Theriogenology 45:817–832[CrossRef][Medline]
  15. Greenwald GS, Roy SK 1994 Follicular development and its control. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 629–724
  16. Vander AJ, Sherman JH, Luciano DS 1994 Reproduction. In: Prancan KM, Bradley JW, eds. Human physiology: the mechanisms of body function. New York: McGraw-Hill; 661–692
  17. Fair T 2003 Follicular oocyte growth and acquisition of developmental competence. Anim Reprod Sci 78:203[CrossRef][Medline]
  18. Johnson AL 2003 Intracellular mechanisms regulating cell survival in ovarian follicles. Anim Reprod Sci 78:185[CrossRef][Medline]
  19. Kobayashi Y, Jimenez Krassel F, Li Q, Yao J, Huang R, Ireland JJ, Coussens PM, Smith GW 2004 Evidence that cocaine- and amphetamine-regulated transcript is a novel intraovarian regulator of follicular atresia. Endocrinology 145:5373–5383[CrossRef][Medline]
  20. Kristensen P, Judge ME, Thim L, Ribel U, Christjansen KN, Wulff BS, Clausen JT, Jensen PB, Madsen OD, Vrang N, Larsen PJ, Hastrup S 1998 Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393:72–76[CrossRef][Medline]
  21. Lambert PD, Couceyro PR, McGirr KM, Dall Vechia SE, Smith Y, Kuhar MJ 1998 CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29:293–298[CrossRef][Medline]
  22. Vrang N, Tang Christensen M, Larsen PJ, Kristensen P 1999 Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour. Brain Res 818:499–509[CrossRef][Medline]
  23. Hunter RG, Philpot K, Vicentic A, Dominguez G, Hubert GW, Kuhar MJ 2004 CART in feeding and obesity. Trends Endocrinol Metab 15:454–459[CrossRef][Medline]
  24. Vrang N, Larsen PJ, Kristensen P, Tang Christensen M 2000 Central administration of cocaine-amphetamine-regulated transcript activates hypothalamic neuroendocrine neurons in the rat. Endocrinology 141:794–801[Abstract/Free Full Text]
  25. Raptis S, Fekete C, Sarkar S, Rand WM, Emerson CH, Nagy GM, Lechan RM 2004 Cocaine- and amphetamine-regulated transcript co-contained in thyrotropin-releasing hormone (TRH) neurons of the hypothalamic paraventricular nucleus modulates TRH-induced prolactin secretion. Endocrinology 145:1695–1699[CrossRef][Medline]
  26. Kuriyama G, Takekoshi S, Tojo K, Nakai Y, Kuhar MJ, Osamura RY 2004 Cocaine- and amphetamine-regulated transcript peptide in the rat anterior pituitary gland is localized in gonadotrophs and suppresses prolactin secretion. Endocrinology 145:2542–2550[CrossRef][Medline]
  27. Baranowska B, Wolinska Witort E, Chmielowska M, Martynska L, Baranowska Bik A 2003 Direct effects of cocaine-amphetamine-regulated transcript (CART) on pituitary hormone release in pituitary cell culture. Neuroendocrinol Lett 24:224–226[Medline]
  28. Smith SM, Vaughan JM, Donaldson CJ, Rivier J, Li C, Chen A, Vale WW 2004 Cocaine- and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism. Endocrinology 145:5202–5209[CrossRef][Medline]
  29. Jaworski JN, Vicentic A, Hunter RG, Kimmel HL, Kuhar MJ 2003 CART peptides are modulators of mesolimbic dopamine and psychostimulants. Life Sci 73:741[CrossRef][Medline]
  30. Kim JH, Creekmore E, Vezina P 2003 Microinjection of CART peptide 55–102 into the nucleus accumbens blocks amphetamine-induced locomotion. Neuropeptides 37:369[CrossRef][Medline]
  31. Kuhar MJ JJ, Hubert GW, Philpot KB, Dominguez G 2005 Cocaine- and amphetamine-regulated transcript peptides play a role in drug abuse and are potential therapeutic targets. AAPS J 7:E259–E265
  32. Ekblad E, Kuhar M, Wierup N, Sundler F 2003 Cocaine- and amphetamine-regulated transcript: distribution and function in rat gastrointestinal tract. Neurogastroenterol Motil 15:545–557[CrossRef][Medline]
  33. Wierup N, Sundler F 2006 CART is a novel islet regulatory peptide. Peptides 27:2031[CrossRef][Medline]
  34. Wierup N, Bjorkqvist M, Kuhar MJ, Mulder H, Sundler F 2006 CART regulates islet hormone secretion and is expressed in the ß-cells of type 2 diabetic rats. Diabetes 55:305–311[Abstract/Free Full Text]
  35. Wierup N, Richards WG, Bannon AW, Kuhar MJ, Ahren B, Sundler F 2005 CART knock out mice have impaired insulin secretion and glucose intolerance, altered ß cell morphology and increased body weight. Regul Pept 129:203–211[CrossRef][Medline]
  36. Vicentic A, Lakatos A, Kuhar MJ 2005 CART (cocaine- and amphetamine-regulated transcript) peptide receptors: specific binding in AtT20 cells. Eur J Pharmacol 528:188–189[CrossRef][Medline]
  37. Gutierrez CG, Campbell BK, Webb R 1997 Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics. Biol Reprod 56:608–616[Abstract]
  38. Jimenez Krassel F, Winn ME, Burns D, Ireland JL, Ireland JJ 2003 Evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by granulosa cells. Endocrinology 144:1876–1886[Abstract/Free Full Text]
  39. Jimenez Krassel F, Ireland JJ 2002 Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production. J Dairy Sci 85:68–78[Abstract]
  40. Li Q, Bakke LJ, Pursley JR, Smith GW 2004 Localization and temporal regulation of tissue inhibitors of metalloproteinases 3 and 4 in bovine preovulatory follicles. Reproduction 128:555–564[Abstract/Free Full Text]
  41. Bettegowda A, Patel OV, Ireland JJ, Smith GW 2006 Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ß-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, ß-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro. Mol Reprod Dev 73:267–278[CrossRef][Medline]
  42. Evans AC, Ireland JL, Winn ME, Lonergan P, Smith GW, Coussens PM, Ireland JJ 2004 Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle. Biol Reprod 70:1475–1484[Abstract/Free Full Text]
  43. Sen A, Choudhary E, Inskeep EK, Flores JA 2005 Effects of selective protein kinase c isozymes in prostaglandin 2 {alpha}-induced Ca2+ signaling and luteinizing hormone-induced progesterone accumulation in the mid-phase bovine corpus luteum. Biol Reprod 72:976–984[Abstract/Free Full Text]
  44. Conti M 2002 Specificity of the cyclic adenosine 3',5'-monophosphate signal in granulosa cell function. Biol Reprod 67:1653–1661[Abstract/Free Full Text]
  45. Parakh TN, Hernandez JA, Grammer JC, Weck J, Hunzicker-Dunn M, Zeleznik AJ, Nilson JH 2006 Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires ß-catenin. Proc Natl Acad Sci USA 103:12435–12440[Abstract/Free Full Text]
  46. Berg JM, Tymoczko JL, Stryer L 2002 Biochemistry. 5th ed. New York: W. H. Freeman and Co.
  47. Burnay MM, Python CP, Vallotton MB, Capponi AM, Rossier MF 1994 Role of the capacitative calcium influx in the activation of steroidogenesis by angiotensin-II in adrenal glomerulosa cells. Endocrinology 135:751–758[Abstract]
  48. Cooke BA 1999 Signal transduction involving cyclic AMP-dependent and cyclic AMP-independent mechanisms in the control of steroidogenesis. Mol Cell Endocrinol 151:25[CrossRef][Medline]
  49. Flores JA, Veldhuis JD, Leong DA 1990 Follicle-stimulating hormone evokes an increase in intracellular free calcium ion concentrations in single ovarian (granulosa) cells. Endocrinology 127:3172–3179[Abstract/Free Full Text]
  50. Babu PS, Jiang L, Sairam AM, Touyz RM, Sairam MR 1999 Structural features and expression of an alternatively spliced growth factor type I receptor for follitropin signaling in the developing ovary. Mol Cell Biol Res Commun 2:21[CrossRef][Medline]
  51. Touyz RM, Jiang L, Sairam MR 2000 Follicle-stimulating hormone mediated calcium signaling by the alternatively spliced growth factor type I receptor. Biol Reprod 62:1067–1074[Abstract/Free Full Text]
  52. Yermolaieva O, Chen J, Couceyro PR, Hoshi T 2001 Cocaine- and amphetamine-regulated transcript peptide modulation of voltage-gated Ca2+ signaling in hippocampal neurons. J Neurosci 21:7474–7480[Abstract/Free Full Text]
  53. Simoni M, Gromoll J, Nieschlag E 1997 The follicle stimulating hormone receptor: biochemistry, molecular biology, physiology and pathophysiology. Endocr Rev 18:739–773[Abstract/Free Full Text]
  54. Miller RJ 1987 Multiple calcium channels and neuronal function. Science 235:46–52[Abstract/Free Full Text]
  55. Patrizio M, Slepko N, Levi G 1997 Opposite regulation of adenylyl cyclase by protein kinase C in astrocyte and microglia cultures. J Neurochem 69:1267–1277[Medline]
  56. Chabardes D, Brick-Ghannam C, Montegut M, Siaume-Perez S 1988 Effect of PGE2 and {alpha}-adrenergic agonists on AVP-dependent cAMP levels in rabbit and rat CCT. Am J Physiol 255:F43–F48
  57. Patel TB, Du Z, Pierre S, Cartin L, Scholich K 2001 Molecular biological approaches to unravel adenylyl cyclase signaling and function. Gene 269:13–25[CrossRef][Medline]
  58. Babu PS, Krishnamurthy H, Chedrese PJ, Sairam MR 2000 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 275:27615–27626[Abstract/Free Full Text]
  59. Hu C-L, Cowan RG, Harman RM, Quirk SM 2004 Cell cycle progression and activation of Akt kinase are required for insulin-like growth factor I-mediated suppression of apoptosis in granulosa cells. Mol Endocrinol 18:326–338[Abstract/Free Full Text]
  60. Evans ACO, Martin F 2000 Kinase pathways in dominant and subordinate ovarian follicles during the first wave of follicular development in sheep. Anim Reprod Sci 64:221[CrossRef][Medline]
  61. Sirotkin AV, Makarevich AV 1999 GH regulates secretory activity and apoptosis in cultured bovine granulosa cells through the activation of the cAMP/protein kinase A system. J Endocrinol 163:317–327[Abstract]
  62. Cameron MR, Foster JS, Bukovsky A, Wimalasena J 1996 Activation of mitogen-activated protein kinases by gonadotropins and cyclic adenosine 5'-monophosphates in porcine granulosa cells. Biol Reprod 55:111–119[Abstract]
  63. Lakatos A, Prinster S, Vicentic A, Hall RA, Kuhar MJ 2005 Cocaine- and amphetamine-regulated transcript (CART) peptide activates the extracellular signal-regulated kinase (ERK) pathway in AtT20 cells via putative G-protein coupled receptors. Neurosci Lett 384:198–202[CrossRef][Medline]
  64. Conti M, Richter W, Mehats C, Livera G, Park J-Y, Jin C 2003 Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J Biol Chem 278:5493–5496[Free Full Text]
  65. Cottom J, Salvador LM, Maizels ET, Reierstad S, Park Y, Carr DW, Davare MA, Hell JW, Palmer SS, Dent P, Kawakatsu H, Ogata M, Hunzicker-Dunn M 2003 Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. J Biol Chem 278:7167–7179[Abstract/Free Full Text]
  66. Platano D, Magli MC, Ferraretti AP, Gianaroli L, Aicardi G 2005 L- and T-type voltage-gated Ca2+ channels in human granulosa cells: functional characterization and cholinergic regulation. J Clin Endocrinol Metab 90:2192–2197[Abstract/Free Full Text]
  67. Asem EK, Qin W, Rane SG 2002 Effect of basal lamina of ovarian follicle on T- and L-type Ca2+ currents in differentiated granulosa cells. Am J Physiol Endocrinol Metab 282:E184–E196
  68. Agoston A, Kunz L, Krieger A, Mayerhofer A 2004 Two types of calcium channels in human ovarian endocrine cells: involvement in steroidogenesis. J Clin Endocrinol Metab 89:4503–4512[Abstract/Free Full Text]
  69. Gao XB, van den Pol AN 2002 Melanin-concentrating hormone depresses L-, N-, and P/Q-type voltage-dependent calcium channels in rat lateral hypothalamic neurons. J Physiol 542:273–286[Abstract/Free Full Text]
  70. Lin Y-F, Tseng M-J, Hsu H-L, Wu Y-W, Lee Y-H, Tsai Y-H 2006 A novel follicle-stimulating hormone-induced G {alpha}h/phospholipase C{Delta}1 signaling pathway mediating rat Sertoli cell Ca2+-influx. Mol Endocrinol 20:2514–2527[Abstract/Free Full Text]
  71. Hunzicker-Dunn M, Maizels ET 2006 FSH signaling pathways in immature granulosa cells that regulate target gene expression: branching out from protein kinase A. Cell Signal 18:1351[CrossRef][Medline]
  72. Xu Y, Zhang W, Klaus J, Young J, Koerner I, Sheldahl LC, Hurn PD, Martinez-Murillo F, Alkayed NJ 2006 Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection. Proc Natl Acad Sci USA 103:14489–14494[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
F. Jimenez-Krassel, J.K. Folger, J.L.H. Ireland, G.W. Smith, X. Hou, J.S. Davis, P. Lonergan, A.C.O. Evans, and J.J. Ireland
Evidence That High Variation in Ovarian Reserves of Healthy Young Adults Has a Negative Impact on the Corpus Luteum and Endometrium During Estrous Cycles in Cattle
Biol Reprod, June 1, 2009; 80(6): 1272 - 1281.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
J.J. Ireland, A.E. Zielak-Steciwko, F. Jimenez-Krassel, J. Folger, A. Bettegowda, D. Scheetz, S. Walsh, F. Mossa, P.G. Knight, G.W. Smith, et al.
Variation in the Ovarian Reserve Is Linked to Alterations in Intrafollicular Estradiol Production and Ovarian Biomarkers of Follicular Differentiation and Oocyte Quality in Cattle
Biol Reprod, May 1, 2009; 80(5): 954 - 964.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
N. Forde, M. Mihm, M. J. Canty, A. E. Zielak, P. J. Baker, S. Park, P. Lonergan, G. W. Smith, P. M. Coussens, J. J. Ireland, et al.
Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle
Physiol Genomics, April 1, 2008; 33(2): 193 - 204.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Bettegowda, J. Yao, A. Sen, Q. Li, K.-B. Lee, Y. Kobayashi, O. V. Patel, P. M. Coussens, J. J. Ireland, and G. W. Smith
JY-1, an oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle
PNAS, November 6, 2007; 104(45): 17602 - 17607.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sen, A.
Right arrow Articles by Smith, G. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sen, A.
Right arrow Articles by Smith, G. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals