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Endocrinology, doi:10.1210/en.2004-0283
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Endocrinology Vol. 145, No. 11 5373-5383
Copyright © 2004 by The Endocrine Society

Evidence that Cocaine- and Amphetamine-Regulated Transcript Is a Novel Intraovarian Regulator of Follicular Atresia

Yasuhiro Kobayashi, Fermin Jimenez-Krassel, Qinglei Li, Jianbo Yao, Ruiping Huang, James J. Ireland, Paul M. Coussens and George W. Smith

Laboratory of Mammalian Reproductive Biology and Genomics (Y.K., Q.L., G.W.S.), Departments of Animal Science (Y.K., F.J.-K., Q.L., J.Y., R.H., J.J.I., P.M.C., G.W.S.) and Physiology (J.Y., J.J.I., G.W.S.), and Center for Animal Functional Genomics (J.Y., J.J.I., P.M.C., 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
 
We recently obtained evidence that cocaine- and amphetamine-regulated transcript (CART), a potent anorectic neuropeptide, is expressed in the bovine ovary. The objectives of this study were to characterize bovine ovarian CART and determine its localization, regulation, and regulatory role during follicular development. CART mRNA was detected in stroma of adult ovaries and in large follicles, but was undetectable in several peripheral tissues, fetal ovaries, and corpora lutea. Within the ovary, CART mRNA and peptide were localized to the granulosal layer of some, but not all, antral follicles, with low, but detectable, expression in oocytes and cumulus cells. CART mRNA was undetectable in granulosal cells of dominant ovulatory follicles collected before and after the preovulatory gonadotropin surge, but was detected in the granulosal layer of adjacent subordinate follicles. In addition, amounts of CART mRNA and follicular fluid concentrations of CART peptide were greater in subordinate follicles vs. dominant follicles of the first follicular wave. Furthermore, CART treatment inhibited basal estradiol production, but not progesterone production, by granulosal cells in a dose-dependent fashion, and the effect was dependent on stage of cell differentiation. We conclude that granulosal cell CART expression is temporally regulated and potentially associated with follicle health status, and CART can inhibit granulosal cell estradiol production. Thus, CART may be a novel local regulator of follicular atresia in the bovine ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN FOLLICLES DEVELOP in a wave-like pattern in many species, including cattle (1, 2, 3), humans (4), and pigs (5). In cattle, each follicular wave lasts 7–10 d, and cattle may have either two or three follicular waves within one estrous cycle (6), with the second or third wave being the ovulatory wave (2). Each follicular wave is preceded by a transient elevation in blood FSH concentrations (3). A cohort of follicles begins to grow at the initiation of each follicular wave. One of the follicles within the cohort continues to grow and becomes the dominant follicle, whereas the rest of the follicles within the cohort become subordinate follicles and undergo atresia. Growth as well as production of estradiol by the dominant follicle are also regulated by LH (7, 8), and the dominant follicle grows to a size similar to that of the preovulatory follicle. However, in the absence of ovulatory signals, the dominant follicle also undergoes atresia. Once the dominant follicle begins to undergo atresia, another cohort of follicles begins to grow, and the cycle of dominant follicle development is repeated.

In addition to the well established regulatory role of pituitary gonadotropins, a growing body of evidence indicates that the development and function of antral follicles are also regulated by locally produced growth factors (9, 10, 11, 12, 13, 14). For example, the oocyte produces two known paracrine growth factors (growth differentiation factor-9 and bone morphogenetic protein-15) that belong to the TGF-ß superfamily, are crucial for normal preantral follicular development (11, 12), and also influence granulosal cell function in antral follicles (10). Local regulation of follicular development has received considerable attention in recent years, and the roles of IGFs (15, 16) and inhibin and activin (12, 17, 18) in the regulation of follicular development and the function of granulosal cells have also been established. However, many additional oocyte- and granulosal cell-derived factors involved in the regulation of follicular development remain undiscovered.

During experiments to characterize the transcriptome of bovine oocytes, we recently obtained expressed sequence tags (ESTs) from a bovine oocyte cDNA library with similarity to human cocaine- and amphetamine-regulated transcript (CART) (19). To our knowledge, the expression of CART mRNA in the mammalian ovary or oocyte has not been previously reported. CART was initially discovered by differential display RT-PCR analysis of brains of rats administered cocaine (20). CART is expressed primarily in the central nervous system or in cells of neuronal origin (21). Within the hypothalamus, CART acts as a potent anorectic peptide and is regulated by leptin (22). The cellular mechanisms that mediate the biological activities of CART are currently unclear, because specific CART receptors have not been identified. However, intracerebroventricular CART infusion can induce c-Fos expression in the hypothalamus and other brain regions (23, 24, 25). In addition, CART acts as a potent inhibitor of L-type voltage-gated calcium channel activity in hippocampal neurons (26).

Based on these findings, we hypothesized that CART may also function as a potential local regulator of follicular development. Our initial objectives, therefore, were to further characterize bovine ovarian CART, determine the tissue distribution of CART mRNA, elucidate the intraovarian localization and temporal regulation of CART mRNA and protein during bovine follicular development, and determine the effect of CART on granulosal cell steroidogenesis. Our results indicate that CART is expressed in the oocyte, cumulus cells, and granulosal cells of antral bovine follicles, and granulosal cell expression is temporally regulated during follicular development and potentially associated with follicle health status. Our results also indicate that CART is a specific inhibitor of basal estradiol production by bovine granulosal cells, but the effects are dependent on the stage of granulosal cell differentiation, suggesting a potential key role for CART in regulation of follicular atresia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care
All animal experiments were approved by the Michigan State University Institutional Animal Care and Use Committee.

RNA isolation and cDNA synthesis
Total RNA was isolated using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Isolated RNA was dissolved in sterile water treated with 0.1% (vol/vol) diethylpyrocarbonate. Before cDNA synthesis, 2.5 µg total RNA were incubated with 2.5 µl 10x deoxyribonuclease (DNase) buffer and 2.5 U DNase I (Invitrogen Life Technologies) at 25 C for 15 min to remove genomic DNA. DNase digestion was terminated by adding 2.5 µl 25 mM EDTA and heating at 65 C for 10 min. The cDNA was synthesized from 1 µg DNase-treated RNA using Superscript II RNase H– reverse transcriptase (Invitrogen Life Technologies), transferred to a sterile screw-cap microcentrifuge tube, and stored at –20 C until use.

Cloning of CART cDNA
The CART EST sequences from the bovine oocyte cDNA library were short and did not contain the entire open reading frame encoding for the CART peptide. Thus, the National Center for Biotechnology Information GenBank EST database was searched to identify bovine EST with similarity to human CART. A bovine EST derived from a mixed tissue cDNA library (GenBank accession no. AW 335960), with sequence similarity to human CART and containing the putative start codon, was identified. A forward primer (5'-ACG CGT CCG GTT TCA GCA CCA T-3') was constructed based on this sequence. A reverse primer (5'-CTT GAC AGA TGA CAT CAC AACC-3') was constructed based on the bovine oocyte EST sequence. Primers were designed based on human CART gene structure to presumably span two introns and amplify a 728-bp cDNA containing the entire coding sequence. Total RNA from bovine hypothalamus (positive control) and stroma (containing small antral follicles) from adult ovary (ovary from sexually mature nonpregnant cow) were reverse transcribed, and respective cDNA were amplified by PCR. The thermal cycler program used consisted of 35 cycles at 94 C for 1 min, 55 C for 1 min, and 72 C for 2 min, with a final extension at 72 C for 10 min. Amplified cDNA encoding for CART of hypothalamic and ovarian origin were ligated into the pBluescript SK+ vector (Stratagene, La Jolla, CA) or pCRII-TOPO vector (Invitrogen Life Technologies). Plasmids containing inserts of interest were then subjected to fluorescent dye terminator sequencing.

Tissue distribution of CART mRNA
Tissue distribution of CART mRNA in various bovine tissues was determined using RT-PCR. Adult and fetal bovine ovaries were collected at a local abattoir. The ages of the fetuses were estimated by measuring crown-rump length (27). Fetal ovaries were collected from animals at approximately d 210 and 260 of gestation (n = 2 each). Ovarian stroma, large antral follicles (>10 mm in diameter), and corpora lutea (CL) were collected from adult ovaries at random stages of the estrous cycle (n = 3 each). Large antral follicles and CL were dissected free of ovarian stroma. Follicular fluid was aspirated and frozen on dry ice. Samples of heart, lung, adrenal, spleen, hypothalamus, and liver were collected after euthanasia of Holstein steers (n = 3) by iv injection of 85 mg/kg sodium pentobarbital. All tissue samples for RNA isolation were frozen in liquid nitrogen and stored at –80 C until RNA isolation. cDNA was synthesized as described above from total RNA isolated from the above tissues. As a positive control, a 314-bp ribosomal protein L-19 (RPL-19) cDNA was amplified from each cDNA sample. PCR conditions for RPL-19 were identical to those for CART. The identity of CART amplicons generated via RT-PCR was determined by agarose gel electrophoresis and Southern analysis (28). A shorter (232-bp) CART cDNA was generated using primers internal to the original PCR primers (forward primer, 5'-CTG GAC ATC TAC TCT GCC GT-3'; reverse primer, 5'-GAA GCG TGG GTG CCT CAT A-3') and radiolabeled with 32P using the random prime labeling procedure (Random Primers DNA Labeling System, Invitrogen Life Technologies). The membrane was incubated with 10 ml ULTRAHyb buffer (Ambion, Inc., Austin, TX) at 42 C for 30 min before hybridization with 32P-labeled probe for 18 h at 42 C. The membrane was then washed in low stringency buffer (29, 30, 31) for 20 min at 50 C, twice in high stringency wash buffer (29, 30, 31) for 20 min each time at 50 and 54 C, and exposed to Hyperfilm MP film (Amersham Biosciences, Piscataway, NJ) at –80 C for 12 h.

Characterization of size of CART mRNA transcripts
The size of CART mRNA transcripts was determined by Northern analysis using previously described procedures (29, 30, 31) with a minor modification. Polyadenylated RNA used was purified from 150 µg total cellular RNA isolated from hypothalamus, ovarian stroma containing small antral follicles, and large follicles (>10 mm in diameter) using a commercially available kit (PolyATract mRNA Isolation System, Promega Corp., Madison, WI) and fractionated in a 1% agarose gel under denaturing conditions. Probe labeling and washing conditions were described above. After washing, the membrane was exposed to a phosphorimager screen for 30 min. The size of the CART RNA transcripts detected was calculated based on comparison with relative migration of 0.24- to 9.5-kb RNA markers (Invitrogen Life Technologies).

Intrafollicular expression of CART mRNA and regulation of expression during the preovulatory period
Samples of stroma of adult bovine ovary (containing ≤3-mm follicles) were collected from ovaries of three different animals at a local abattoir, embedded in embedding medium (Tissue-Tek, Sakura Fintek U.S.A., Inc., Torrance, CA), frozen over liquid nitrogen vapors, and transported to the laboratory on dry ice. The collection of ovarian tissues containing dominant ovulatory follicles and adjacent subordinate follicles has been reported previously (29, 30, 31). Samples collected at 0 and 12 h (n = 3 each) after GnRH injection were used.

The expression of CART mRNA within ovarian tissues (ovarian stroma containing small antral follicles and dominant ovulatory follicles with adjacent subordinate follicles collected at 0 or 12 h post-GnRH injection) was examined using in situ hybridization as described previously (29, 30, 31), but with the following modification. Washing of slides after ribonuclease A digestion was performed at 65 C. After washing, slides were exposed to autoradiographic emulsion for 7 d at 4 C before development, counterstaining, and acquisition of digital dark- and bright-field images. Ten serial sections from each sample were examined.

RT-PCR experiments were also performed to conclusively demonstrate the presence of CART mRNA in isolated oocytes and cumulus cells. Germinal vesicle stage oocytes were collected via aspiration of 3- to 6-mm bovine follicles from ovaries collected at an abattoir. Six pools of grade I oocytes (10 oocytes/pool) and matching cumulus cells were generated. Oocytes were completely denuded of cumulus cells, and RNA was isolated from oocytes and cumulus cells for each sample using the RNAqueous Micro Scale RNA isolation kit (Ambion, Inc.). RT-PCR was conducted as described above.

Immunohistochemical localization of the CART peptide
Samples of stroma of adult ovary (containing ≤3-mm follicles) were collected at a local abattoir from ovaries of three different animals at random stages of the estrous cycle. Samples were placed in a plastic tissue cassette (Histosette II, Fisher Scientific Co., Pittsburgh, PA), fixed in 10% (vol/vol) neutral buffered formalin, and embedded in paraffin. Immunohistochemical localization of the CART peptide was performed using previously described procedures (32) and rabbit antirat CART (55–102) polyclonal antisera (Phoenix Pharmaceuticals, Inc., Belmont, CA) at a 1:1000 dilution. Parallel controls were used, including sections incubated with a similar dilution of normal rabbit serum or rabbit anti-CART serum that had been preincubated overnight at 4 C with 10 µg/ml rat CART (55–102) peptide (American Peptide Co., Sunnyvale, CA). Ten serial sections from each sample were examined.

Differential expression of CART mRNA and the CART peptide in first wave dominant vs. subordinate follicles
Ovaries bearing follicles from the first wave of follicular development were collected at a local abattoir based on the appearance of the corpus luteum as previously described (33, 34). The largest (dominant) and second largest (subordinate) follicles (n = 5 each) were dissected free of stroma, and the diameter of each follicle was measured using a caliper (average diameter of dominant follicles, 13.5 ± 0.73 mm; average diameter of subordinate follicles, 6.9 ± 1.07 mm). Follicular fluid was aspirated from each follicle, and an aliquot (60 µl) from each sample was treated with 10 µl 7x protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). Follicular fluid was frozen on dry ice, and follicular tissue was frozen in liquid nitrogen. Samples were stored at –80 C until use. Concentrations of 17ß-estradiol (E) and progesterone (P) in follicular fluid samples were measured by respective RIAs (Diagnostic Products Corp., Los Angeles, CA) (34). Inter- and intraassay coefficients of variation (CV) for E assay were 7% and 6%, respectively. Intrafollicular concentrations of P were measured in a single assay. The intraassay CV was 8.7%. The health status of each follicle was determined by calculating the ratio between E and P concentrations. Follicles with an E:P ratio of 1 or greater were considered estrogen active (EA), whereas follicles with E:P ratio less than 1 were considered estrogen inactive (EI). EA follicles have biochemical characteristics of healthy growing follicles, whereas EI follicles are destined to undergo atresia (35, 36, 37).

Real-time RT-PCR was used to quantify amounts of CART mRNA in first wave dominant and subordinate follicles. Total RNA from dominant follicles with an E:P ratio greater than 1.0 and subordinate follicles with an E:P ratio less than 1 were used for analyses (n = 5 each). Synthesis of cDNA was performed as described above. Primers were designed using the Primer Express program (Applied Biosystems, Foster City, CA) and were derived from the bovine CART nucleotide sequence obtained above. The PCR mixture contained 50 ng cDNA, 25 µl SYBR Green PCR Master Mix (Applied Biosystems), 45 pM forward primer (5'-TGT GAC TGT CCC CGA GGA A-3'), and 15 pM reverse primer (5'-GAA GCG TGG GTG CCT CAT A-3') in a total reaction volume of 50 µl. As an internal control, the amount of ß-actin mRNA in each sample was quantified as described above, except that 15 pM of each primer was used (forward primer, 5'-CGC CAT GGA TGA TGA TAT TGC-3'; reverse primer, 5'-AAG CCG GCC TTG CAC AT-3'). Standard curves for CART and ß-actin were produced from their respective PCR products after purification (QIAquick PCR purification kit, Qiagen, Valencia, CA). The amounts of PCR product used were 100 ag, 1, 10, and 100 fg, and 1 and 10 pg for CART and ß-actin standard curves. Reactions were performed in duplicate for each sample in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The thermal cycler program consisted of 40 cycles of 95 C for 15 sec and 60 C for 1 min. The amounts of CART and ß-actin mRNA in each sample were determined using ABI PRISM Sequence Detection System software (Applied Biosystems) by comparison of cycle threshold for each sample with that of respective standard curve. The CART standard curve had a slope of –3.277 (r2 = 0.998). The ß-actin standard curve had a slope of –3.37 (r2 = 0.992).

A commercially available RIA kit for rat CART (55–102) (Phoenix Pharmaceuticals) was validated for measuring concentrations of the CART peptide in bovine follicular fluid. Follicular fluid samples from above EA dominant and EI subordinate follicles were used (n = 4 each). Samples of follicular fluid were diluted in RIA buffer, and concentrations were determined in duplicate in a single RIA according to manufacturer’s instruction. The intraassay CV was 12.2%. Increasing amounts of diluted follicular fluid samples (10, 25, 50, and 100 µl) produced a displacement curve parallel to the standard curve. The addition of increasing amounts of CART (8, 16, 32, and 64 pg/tube) to a fixed volume of follicular fluid (50 µl) yielded an average 99.1% recovery. The assay sensitivity was 1 pg/tube. According to the manufacturer, CART (55–102) antisera used in the RIA displays no cross-reactivity with other hormones/peptides, including leptin, orexin, agouti-related protein, and neuropeptide Y.

Effects of CART on steroidogenesis of cultured granulosal cells
The effects of CART on in vitro production of E and P were determined using a short-term granulosal cell culture system described previously (34, 38). Granulosal cells were harvested from the largest follicle from random stages of the first wave of follicular development (n = 8 follicles) as described above. Granulosal cells were plated in triplicate at 100,000 cells/well and cultured for 18 h in the presence of 0, 0.01, 0.1, or 0.5 µM rat CART (55–102) peptide (American Peptide). At the end of culture, granulosal cells were examined to determine whether incubation with CART affected cell morphology. Culture medium from each well was harvested, and concentrations of E and P were measured using commercially available RIA kits as previously described (34, 38). The inter- and intraassay CVs for E were 7% and 6%, respectively. The inter- and intraassay CVs for P were 9.8% and 4.2%, respectively.

Statistical analyses
The effect of stage of follicular development on amounts of CART mRNA (femtograms) and ß-actin mRNA (picograms) was analyzed using the general linear model procedure of SAS (version 8, SAS Institute, Cary, NC). Amounts of CART mRNA were normalized relative to ß-actin mRNA, and data were log-transformed before analyses. The model included follicle (dominant vs. subordinate) as the main effect. The effect of stage of follicular development on intrafollicular CART peptide concentrations was also analyzed using the general linear model procedure of SAS. Data were log-transformed before analyses. The model included follicle (dominant vs. subordinate) as the main effect. Data are shown as the least square mean ± SE.

Concentrations of E and P in culture medium of CART-treated cells were converted to the percentage of untreated controls (cells treated with 0 µM rat CART) to account for differences in basal E and P production across different follicles and analyzed using the general linear model of SAS. The model included doses of CART (0, 0.01, 0.1, or 0.5 µM) as the main effect. Means were separated using Tukey’s test. Changes in production of E and P in relation to different doses of CART were analyzed using the regression procedure of SAS to test for a significant dose response. The model included doses of CART (0, 0.01, 0.1, or 0.5 µM) as the main effect.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of bovine CART cDNA from ovarian and hypothalamic tissues and characterization of CART mRNA
The nucleotide sequence of the bovine CART cDNAs derived from hypothalamic and ovarian RNA was identical (data not shown). The nucleotide sequence of bovine CART shared 85% and 90% homology with that of rat and human CART, respectively (data not shown). The predicted amino acid sequence of the bovine hypothalamic and ovarian CART peptide was also highly similar to human (97%) and rat (86%) CART (Fig. 1AGo). As observed for human CART, 13 amino acids found within the predicted amino acid sequence of rat CART were absent in the putative bovine CART peptide sequence. A small number of amino acid substitutions were found between bovine CART and human CART (six amino acids) as well as between bovine and rat CART (three amino acids). The majority of amino acid substitutions observed were conservative and found within the signal peptide (Fig. 1AGo). There was a valine to isoleucine substitution in the N terminus of the biologically active region (amino acids 55–102) of human vs. bovine or rat CART (Fig. 1AGo). However, the predicted amino acid sequence of the core region of the biologically active CART peptide (amino acids 61–102) was identical among species compared (Fig. 1AGo).



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  		FIG. 1. Characterization of bovine CART mRNA. A, Comparison of predicted amino acid sequence of bovine hypothalamic and ovarian CART with rat and human CART. {cjs2112}, Location of the signal peptide; {square}, location of the biologically active portion of the CART peptide [CART (55–102)]; {blacksquare}, core region of CART (61–102). Amino acid substitutions in rat and human CART vs. bovine CART are denoted in bold. The dotted line denotes the location of the additional 13 amino acids missing from the propiece of bovine and human CART vs. rat CART. B, Northern analysis of bovine CART mRNA. The size and number of CART mRNA transcripts were characterized by Northern analysis using polyadenylated RNA isolated from hypothalamus, adult ovary (containing small antral follicles; denoted as Ovary), and large follicles (>10 mm in diameter; denoted as Follicle).

 
To further characterize bovine CART mRNA, the size of CART mRNA transcripts in bovine ovarian tissues was determined by Northern analysis. The radiolabeled bovine CART cDNA hybridized to a predominant transcript of similar size (~1.0 kb) in bovine hypothalamus, ovarian stroma (adult), and antral follicle RNA samples (Fig. 1BGo). Specific binding to additional minor transcripts was not detected.

Tissue distribution of CART mRNA
A single CART PCR product (728 bp) was detected in cDNA from hypothalamus, but was low or undetectable in cDNA from heart, lung, adrenal, spleen, and liver (Fig. 2Go). Within ovarian tissues, CART mRNA was detected in samples of stroma of adult ovaries, which contained small antral follicles, and in large antral follicles. The expression of CART mRNA was undetectable in CL samples and fetal ovaries (containing primarily preantral follicles) collected at 210 and 260 d gestation. In comparison, a 314-bp cDNA encoding for RPL-19 (positive control) was amplified from all samples. Southern analysis confirmed the identity of all CART amplicons generated (data not shown).



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  		FIG. 2. Tissue distribution of CART mRNA. Representative ethidium bromide-stained gel of RT-PCR detection of CART mRNA in various bovine tissues. cDNA was synthesized from RNA isolated from hypothalamus (HYP), heart, lung, adrenal, spleen, liver, adult ovary (containing small antral follicles; denoted as Ovary), and fetal ovaries (FO) collected at 210 and 260 d gestation, large antral follicles (>10 mm in diameter; denoted as Follicle), and CL and analyzed using RT-PCR. The expression of ribosomal protein L-19 mRNA was used as a positive control. RT (–), Negative control reaction incubated in the absence of reverse transcriptase.

 
Intraovarian expression of CART mRNA and protein
The exact ovarian cell types that express CART mRNA were initially investigated using in situ hybridization. In three samples of adult ovary (containing small antral follicles), CART mRNA was localized within some, but not all, antral follicles (Fig. 3Go, A–D). Within follicles with detectable CART mRNA, expression was clearly localized to the granulosal cell layer (Fig. 3Go, C and D). In comparison, the expression of CART mRNA was not detectable in the thecal cell layer (Fig. 3Go, C and D). Weak, but detectable, localization of CART mRNA to the oocyte was also observed by in situ hybridization (data not shown). Six oocyte-cumulus cell complexes were found in over 30 sections examined, and weak CART mRNA expression was detected in all oocytes examined. However, to confirm oocyte expression of CART mRNA, RT-PCR experiments were performed (Fig. 3FGo). Of the six samples of germinal vesicle stage oocyte RNA examined (10 oocytes/sample), CART mRNA was detected in five of the six samples (Fig. 3FGo). The expression of RPL-19 mRNA (positive control) was detectable in all samples (Fig. 3FGo). Although low to undetectable by in situ hybridization analysis (data not shown), the expression of CART mRNA (as determined by RT-PCR) was also noted in all six cumulus cell RNA samples (Fig. 3FGo). Thus, low, but detectable, levels of CART mRNA are present in the oocyte and cumulus cells of bovine antral follicles.



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  		FIG. 3. Cell-specific expression of CART mRNA within the bovine ovary. A, Representative bright-field micrograph of section through stroma of adult ovary hybridized with [35S]antisense CART cRNA. B, Dark-field image of A. Note the prominent localization of CART mRNA to the granulosa cell layer (GC) of the follicle located at the top of A and B, but not to the GC of the follicle at the bottom of the same panels. C and D, Magnified bright-field (C) and dark-field (D) micrographs illustrating localization of CART mRNA specifically to the GC of the follicle located in the top portion of A. E, Representative dark-field micrograph of a section adjacent to that depicted in A and hybridized with [35S]sense CART cRNA. A and B, Magnification, x100; scale bar, 200 µm. C–E, Magnification, x400; scale bar, 50 µm. GC, Granulosal cell layer; TC, thecal cell layer. F, Ethidium bromide-stained gel of RT-PCR detection of CART mRNA. cDNA was synthesized from RNA isolated from germinal vesicle stage oocytes (n = 6 samples, 10 oocytes/sample) and corresponding cumulus cells. The expression of ribosomal protein L-19 mRNA was used as a positive control. RT (–), Negative control reaction incubated in the absence of reverse transcriptase. Note the detection of CART mRNA in five of six oocyte RNA samples and in all six cumulus cell RNA samples.

 
The intraovarian localization of the CART peptide was determined using immunohistochemistry (Fig. 4Go). Consistent with the pattern of mRNA localization, prominent CART immunoreactivity was localized to the granulosal layer of some (Fig. 4AGo), but not all, antral follicles (data not shown). CART immunoreactivity was also localized to the oocyte and cumulus cells, but with diffuse, weaker staining than observed in the granulosal cell layer (Fig. 4DGo). A total of seven cumulus-oocyte complexes were identified in all sections, and CART immunoreactivity was observed in all cumulus-oocyte complexes examined. Significant immunoreactivity in the granulosal cells, cumulus cells, and oocyte was not detected when adjacent sections were incubated with normal rabbit serum (Fig. 4Go, B and E) or when the CART antiserum was preabsorbed with excess CART peptide (Fig. 4Go, C and F).



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  		FIG. 4. Immunohistochemical localization of the CART peptide within the bovine ovary. A, Representative micrograph of section through stroma of adult bovine ovary incubated with rabbit anti-CART serum. Note the prominent localization of CART immunoreactivity to the granulosal layer of an antral follicle. Inset, Magnified micrograph of selected region from A containing granulosal cells. B, Micrograph of adjacent section (to that depicted in A) incubated with normal rabbit serum. Inset, Magnified micrograph of selected region from B containing granulosal cells. C, Micrograph of adjacent section (to that depicted in B) incubated with rabbit anti-CART serum preabsorbed with an excess of CART peptide. Inset, Magnified micrograph of selected region from C containing granulosal cells. D, Representative micrograph of section (containing oocyte and cumulus cells) through stroma of adult bovine ovary incubated with rabbit anti-CART serum. Note localization of CART immunoreactivity to oocyte and cumulus cells. E, Micrograph of adjacent section (to that depicted in D) incubated with normal rabbit serum. F, Micrograph of adjacent section (to that depicted in E) incubated with rabbit anti-CART serum preabsorbed with an excess of CART peptide. GC, Granulosal cell layer; TC, thecal cell layer; OO, oocyte; CC, cumulus cell layer. A–F: Magnification, x400; scale bar, 50 µm. Insets: Magnification, x1000.

 
Regulation of CART mRNA during the preovulatory period
Regulation of CART mRNA in dominant ovulatory vs. subordinate follicles collected during the preovulatory period was examined by in situ hybridization analysis of ovarian tissue samples collected before and 12 h after GnRH injection to induce a gonadotropin surge (n = 3 each). The expression of CART mRNA was undetectable in dominant ovulatory follicles collected both before (Fig. 5Go, A and C) and after (Fig. 5Go, B and D) the preovulatory gonadotropin surge. However, CART mRNA was clearly localized to the granulosal cell layer of adjacent subordinate (nonovulatory) follicles (Fig. 5Go, A–H).



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  		FIG. 5. In situ localization of CART mRNA within dominant ovulatory vs. nonovulatory (subordinate) bovine follicles collected before (0 h) and after (12 h) GnRH injection to induce the preovulatory gonadotropin surge (n = 3 each). A, Bright-field micrograph of section through dominant ovulatory follicle (DF) and adjacent nonovulatory subordinate follicle (SF) collected before the preovulatory gonadotropin surge (0 h) and hybridized with [35S]antisense CART cRNA. B, Bright-field micrograph of section through DF and adjacent SF collected after the preovulatory gonadotropin surge (12 h) and hybridized with [35S]antisense CART cRNA. C, Darkfield micrograph of A. D, Dark-field micrograph of B. Note localization of CART mRNA to the granulosa cells of the SF (right), but not the DF (left), depicted in A–D. E, Magnified bright-field micrograph of SF depicted in A and C. F, Magnified bright-field micrograph of SF depicted in B and D. G, Magnified dark-field micrograph of E. H, Magnified dark-field micrograph of F. Note localization of CART mRNA specifically to granulosa cells of SF in G and H. I and J, Dark-field micrographs of sections adjacent to those depicted in E and F hybridized with [35S]sense CART cRNA (negative control). A–D, Magnification, x100; scale bar, 200 µm. E–J, Magnification, x400; scale bar, 50 µm. GC, Granulosal cell layer; TC, thecal cell layer.

 
Differential expression of CART mRNA and CART peptide during the first follicular wave
To further determine the association of CART expression with stage of follicular growth and development, the differential regulation of CART mRNA and CART peptide concentrations in first wave dominant and subordinate follicles (collected at a slaughterhouse and from random stages of the wave) was examined (n = 5 each). As expected, intrafollicular concentrations of E were greater in EA dominant follicles compared with EI subordinate follicles (P < 0.05; Fig. 6AGo). However, amounts of CART mRNA were less (P < 0.05) in EA dominant follicles compared with EI subordinate follicles (Fig. 6BGo). The EA dominant and EI subordinate follicles had similar levels of ß-actin mRNA (P > 0.1). Concentrations of CART peptide in follicular fluid samples from EI subordinate follicles were also greater (P < 0.05) than those in EA dominant follicles (Fig. 6CGo).



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  		FIG. 6. Expression of CART mRNA and follicular fluid concentrations of CART peptide in estrogen-active dominant follicles (DF) and estrogen-inactive subordinate follicles (SF) collected at random stages of the first follicular wave. A, Intrafollicular concentrations of E in estrogen-active DF and estrogen-inactive SF collected at random stages of the first follicular wave (n = 5 each; least square mean ± SEM). *, P < 0.05. B, Relative abundance of CART mRNA in estrogen-active DF and estrogen-inactive SF collected at random stages of the first follicular wave (n = 5 each). The expression of CART mRNA was normalized relative to concentrations of ß-actin mRNA and was expressed as the ratio of femtograms of CART mRNA to picograms of ß-actin mRNA (least square means ± SEM). The expression of CART mRNA was increased in estrogen-inactive SF relative to estrogen-active DF. *, P < 0.05. C, Intrafollicular concentrations of CART peptide in estrogen-active DF and estrogen-inactive SF collected at random stages of the first follicular wave (n = 4 each; least square mean ± SEM). The concentrations of CART peptide were also greater in follicular fluid of estrogen-inactive SF relative to estrogen-active DF. *, P < 0.05.

 
Effects of CART on production of E and P by cultured granulosal cells
Addition of rat CART (55–102) peptide to cultured granulosal cells inhibited basal E production in a dose-dependent fashion (P < 0.05) if granulosal cells had high in vivo (>50 ng E/ml in follicular fluid) and in vitro (>400 pg/ml) capacity to produce E (Fig. 7AGo). The response was maximal at 0.1 µM (>30% decrease vs. control; P < 0.05), and basal production of E was not further inhibited in the presence of higher CART concentrations (0.5 µM; P > 0.10 vs. 0.1 µM; data not shown). Addition of CART had no effect (P > 0.10) on in vitro production of E if granulosal cells had low in vivo (<5 ng/ml E in follicular fluid) and in vitro (<400 pg/ml) capacity to produce E (Fig. 7BGo). After 18 h of culture, morphology of granulosal cells treated with different doses of CART appeared similar to that of granulosal cells cultured without CART. The effects of CART on granulosal cell steroidogenesis were specific to E production as CART treatment had no effect on basal P production by granulosal cells at either stage of differentiation (P > 0.10; Fig. 7Go, C and D).



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  		FIG. 7. Effect of CART on granulosal cell steroidogenesis. A, Concentrations of E in culture medium of granulosa cells from follicles (n = 4) with high in vivo (>50 ng E/ml follicular fluid) and in vitro (>400 pg/ml) capacity to produce E that were treated with increasing concentrations (0, 0.01, and 0.1 µM) of CART. Concentrations of E in culture medium decreased in a dose-dependent manner (P < 0.05). The response was maximal at 0.1 µM CART (*, P < 0.05 vs. untreated control), and basal production of E was not further inhibited in the presence of higher CART concentrations (0.5 µM; data not shown). B, Concentrations of E in culture medium of granulosal cells from follicles (n = 4) with low in vivo (<5 ng E/ml follicular fluid) and in vitro (<400 pg/ml) capacity to produce E that were treated with increasing concentrations (0, 0.01, and 0.1 µM) of CART (P > 0.10). C, Concentrations of P in culture medium of granulosal cells from follicles with high in vivo (>50 ng E/ml follicular fluid) and in vitro (>400 pg/ml) capacity to produce E that were treated with increasing concentrations (0, 0.01, and 0.1 µM) of CART (P > 0.10). D, Concentrations of P in culture medium of granulosal cells from follicles with low in vivo (<5 ng E/ml follicular fluid) and in vitro (<400 pg/ml) capacity to produce E that were treated with increasing concentrations (0, 0.01, and 0.1 µM) of CART (P > 0.10). Data are expressed as a percentage of the untreated control value (cells incubated in the absence of CART) to account for differences in basal E and P production. Each bar represents the mean ± SEM percentage of the untreated control value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although our understanding of the endocrine control of antral follicle growth and development is well established, increasing attention in recent years has been focused on both identification and the subsequent contribution of locally produced regulatory molecules to the control of antral follicle growth and development. Results of the present studies clearly demonstrate a low level of expression of the previously described anorectic neuropeptide CART in the oocyte and cumulus cells of antral follicles in the bovine ovary and prominent expression of CART in the granulosal cells. Granulosal cell CART expression was temporally regulated during antral follicle growth and potentially associated with follicle health status. Furthermore, results of the present studies indicate that CART can inhibit E production by bovine granulosal cells, but the effect of CART is dependent on stage of differentiation. Collectively, results presented to date established a novel biological activity for the CART peptide outside of the nervous system and support a potential local regulatory role for CART in follicular atresia.

The amino acid sequence of the CART peptide was first reported for sheep, where it was classified as a somatostatin-like peptide in sheep hypothalamus (39), but its identity remained unknown until 1995, when Douglass and co-workers (20) identified CART as a mRNA species whose expression increased acutely after psychomotor stimulant administration. The nucleotide and predicted amino acid sequence of CART from rat (20), human (40), mouse (41), and sheep (42) have been reported previously and were highly homologous to bovine ovarian CART. However, the expression of CART mRNA in the mammalian ovary has not been reported previously. It is interesting to note that the predicted amino acid sequence of the biologically active region of bovine CART is identical to that of rat CART, but the 13 amino acids that are absent in human (40) and sheep (42) CART are also absent in bovine CART. Furthermore, the size of the predominant bovine CART mRNA transcript is similar to that of human CART mRNA (40). In rat hypothalamus and brain, 700- and 900-bp CART mRNA transcripts are present (20, 41). These transcripts arise from the use of two different polyadenylation sites along with alternative splicing of 39 nucleotides within the coding region. Both potential polyadenylation sites are also present in the nucleotide sequence of bovine CART. However, we did not detect multiple CART mRNA transcripts by Northern analysis of polyadenylated RNA isolated from bovine hypothalamus or ovarian tissues. Douglass and Daoud (40) also did not detect multiple CART mRNA transcripts in RNA isolated from different regions of human brain.

Aside from the brain, CART expression has been detected previously in many tissues, including pituitary gland, adrenal gland (21, 43, 44), stomach (44), and intestines (44, 45). However, within above somatic tissues, the expression of CART is localized in either tissue of neural origin, such as the posterior pituitary gland (43, 44) and adrenal medulla (43) or in nerves that innervate such tissues, including the vagus nerves (25, 46) and myenteric plexus neurons (45). To our knowledge, the expression of CART in somatic cells and germ cells of the mammalian gonad has not been reported previously. Murphy et al. (44) did not detect immunoreactive CART peptide in rat ovaries and testis. Within gonadal tissues, CART expression has been documented in the goldfish ovary (47) and in nerves that innervate the epididymis of rat testis (48). Although mammalian ovaries are innervated (49, 50), our studies clearly showed that both CART mRNA and protein are expressed in the oocyte, cumulus cells, and granulosal cells of antral follicles, none of which are of neural origin or innervated.

Our results also demonstrated that CART expression is temporally regulated during follicular development. The expression of CART was not detected in preantral follicles from fetal or adult bovine ovaries, but was readily detected in antral follicles at various stages of development. These results suggest that the signals and pathways that initiate CART expression in the oocyte and granulosal cells are potentially functional only at the antral stage of follicular development. Furthermore, CART expression was detected in the granulosal cell layer of some, but not all, antral follicles. During follicular development, granulosal cells undergo extensive morphological and functional changes (9, 14). The temporal expression of CART mRNA in granulosal cells of antral, but not preantral, follicles suggests that CART mRNA expression is restricted to granulosal cells that have undergone advanced stages of differentiation. The exact mechanisms that are responsible for the expression of CART mRNA in antral, but not preantral, follicles are unknown.

Although granulosal cell CART expression is restricted to antral follicles, evidence from the first follicular wave and the ovulatory wave in cattle clearly supports a potential association of CART expression with follicle health status and enhanced CART expression in subordinate atretic follicles. During the preovulatory period, abundant expression of CART mRNA was localized to the granulosal cells of nonovulatory subordinate follicles, but CART mRNA was not readily detectable in the granulosal layer of dominant ovulatory follicles collected before and after the preovulatory gonadotropin surge. Health status of above follicles examined was clearly documented by ultrasonography during the follicular wave leading up to sample collection (29). Furthermore, CART mRNA and follicular fluid concentrations of CART peptide were also lower in EA dominant follicles vs. EI subordinate follicles (collected at random stages of the first follicular wave). Ireland and Roche (35, 37) and Sunderland et al. (36) showed that bovine EA follicles (E:P ratio, ≥1.0) had biochemical characteristics of healthy growing follicles and had a lower incidence of atresia compared with EI follicles with low E:P ratio (<1.0). Jolly et al. (51) reported that apoptosis (as assessed by degree of oligonucleosome formation) is much more prominent in granulosal cells collected from EI vs. EA antral bovine follicles. We acknowledge that E:P ratios are an indirect indicator of follicle health status, and additional studies will be required to determine a direct relationship between CART expression and follicle health status (apoptosis). However, in vitro experiments also demonstrated a negative role for CART in the regulation of E production. Thus, the results of the present studies support a potential association of CART expression with follicular atresia. Whether CART functions solely as a negative regulator of E production or also promotes downstream components of the atresia process (apoptosis) will require further investigation.

The development and function of antral follicles are regulated by the complex interaction between endocrine factors and locally produced growth factors (10, 11, 12). Several lines of evidence from studies of cultured rat granulosal cells suggest that neuropeptides such as GnRH, GHRH, and vasoactive intestinal peptide can regulate granulosal cell functions, including steroidogenesis and cAMP synthesis (52, 53, 54). However, the physiological role of such peptides in vivo in regulation of granulosal cell steroidogenesis is unclear. Our results indicate that the previously described neuropeptide CART is locally produced during bovine follicular development and can regulate granulosal cell E production. Intraovarian localization and evidence of a regulatory role for CART during ovarian follicular development have not been reported previously.

Our results clearly demonstrated an inhibitory effect of CART on in vitro production of E by granulosal cells. The concentrations of CART used are potentially physiological and mimic those reported to inhibit L-type voltage-gated calcium channels in hippocampal neurons (26). L-Type voltage-gated calcium channels are present on granulosal cells of rats (55), pigs (56), and birds (57). However, the inhibitory effect of CART on in vitro production of E was not observed after treatment of granulosal cells with low basal E production in vitro and isolated from follicles with low follicular fluid E concentrations. The varying response of granulosal cells of different E-producing capacity to CART stimulation implies that stage of differentiation may be important in determining the responsiveness of cells to the inhibitory effects of CART. Furthermore, granulosal cells used in cell culture experiments were all obtained from an abattoir and collected at random stages of the first follicular wave. Thus, the specific stage of follicular development when granulosal cells are maximally responsive to the inhibitory effects of CART cannot be inferred from the present studies. However, CART treatment of cultured granulosal cells at both stages of differentiation had no effect on basal P production, indicating that the inhibitory effects of CART on granulosal cell steroidogenesis are specific to E production. The exact mechanisms and intracellular signaling pathways that control responsiveness of granulosal cells to CART and mediate the negative effects of CART on E production are unknown, because a specific receptor for CART has not been described to date.

In summary, the results of the present studies have established that CART mRNA and the CART peptide are expressed within the oocyte, cumulus cells, and granulosal cell layer of antral, but not preantral, follicles of the bovine ovary, and CART expression is temporally regulated during antral follicle development. Furthermore, evidence to date indicates that CART expression is potentially associated with follicle health status and that CART can inhibit E production by granulosal cells isolated from healthy EA follicles. Collectively, these results strongly support a potential role for CART in the atresia of antral follicles. Elucidation of the mechanisms that regulate intraovarian CART expression and the precise requirement of CART in vivo for atresia of bovine antral follicles will require further investigation.


   Acknowledgments
 
The authors thank A. Bettegowda for collection of oocytes and cumulus cells.


   Footnotes
 
This work was supported by the Rackham Foundation, the Michigan State University Office of the Vice President for Research and Graduate Studies, and the Michigan Agricultural Experiment Station.

Current address of J.Y.: Division of Animal and Veterinary Science, West Virginia University, Morgantown, West Virginia 26506.

Abbreviations: CART, Cocaine- and amphetamine-regulated transcript; CL, corpora lutea; CV, coefficient of variation; DNase, deoxyribonuclease; E, 17ß-estradiol; EA, estrogen active; EI, estrogen inactive; EST, expressed sequence tag; P, progesterone.

Received March 4, 2004.

Accepted for publication July 13, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals