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AgResearch Ruakura (B.S., L.H., P.L.P.), Hamilton 2001, New Zealand; and University of Auckland, Research Center in Reproductive Medicine, Department of Obstetrics and Gynecology, National Women’s Hospital (A.N.S.), Auckland 1001, New Zealand
Address all correspondence and requests for reprints to: Dr. Peter L. Pfeffer, AgResearch Ruakura, Private Bag 3123, East Street, Hamilton 2001, New Zealand. E-mail: peter.pfeffer{at}agresearch.co.nz.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thus, whereas the central role of FSH in follicle growth has been established, it is far from clear how the future DF is selected to grow under nadir FSH concentrations, whereas other cohort members succumb. One report has documented the emergence of the future DF 6 h before the remainder of the cohort (12). This suggests that the future DF possesses an early growth advantage possibly due to an inherently (stochastically) better developmental potential (e.g. number of FSH receptors) or a more favorable positioning in relation to the blood supply. In the first case the follicle would be able to respond more rapidly to the rise in FSH; in the second it would be exposed earlier to increasing levels of FSH than other follicles in the cohort. The subtle size difference before deviation/selection allows one follicle to rapidly establish dominance (12, 13, 14), but how is this achieved? In principle, the necessary decrease in dependency on FSH, allowing selective survival, can be achieved by an increased sensitivity to low levels of FSH and/or increased dependency on other endo-, para-, or autocrine signals. Factors and pathways that have been proposed to be involved in the selection process thus include estradiol, LH, inhibin, and IGFs.
Around the critical period when deviation/selection occur, the concentration of estradiol is higher in the follicular fluid of the DF than in the SF (12, 15, 16, 17, 18, 19). This is reflected by studies examining follicular expression of cytochrome p450 aromatase, the enzyme required for estradiol synthesis from androstenedione (20, 21). Estrogens are able to amplify the overall effects of FSH (22), thus making them a good candidate for a critical selection factor, particularly in view of recent data that increased estrogen levels in DF slightly precede selection/deviation (18).
The estrogen- and inhibin mediated systemic shift from FSH- to LH-dominated gonadotropin production would suggest that a timely ability to synthesize LH receptors may underlie DF selection as well as survival. Consistent with this idea, LH levels increase 1 d before selection (4). LH receptor mRNA was elevated in the DF 1 d after selection (23) as well as approximately 8 h (
8 mm size) before selection (16, 21). Thus, both the presence of the signaling factor and its receptor are consistent with LH being involved in follicle selection.
The role of the TGFß superfamily members inhibin and activin in follicle selection is less clear due to the large number of posttranslationally processed and monomeric and dimeric combinations of the products of the three inhibin genes and the existence of the activin inhibitor follistatin. Follicular fluid concentrations of free activin and the higher molecular weight precursor forms of inhibin increased, whereas the 34-kDa fully processed inhibin remained constant during the time leading up to selection. However, no significant difference in concentration of these factors could be detected among the three largest follicles around the time of selection (17, 18).
There is increasing evidence that the IGF system, consisting of IGF-I and -II, IGF receptors, a family of six binding proteins (IGFBP), and IGFBP proteases, plays a role in DF selection (1, 2, 4). Apart from stimulating cell proliferation and estradiol production, IGFs enhance the sensitivity of granulosa cells to FSH (24), thus heightening the response of the future DF to the decreasing levels of FSH occurring at selection. Interestingly, the follicular fluid concentration of free IGF-I was lower in subordinate follicles well before selection (16, 18). The increased bioavailability of IGF-I in future DF may be linked to a reduction in the follicular fluid of the three low molecular weight IGFBP (-2, -4, and -5) observed before (14, 25) and after selection (17, 26, 27). One of the reasons for lowered IGFBP-4 and -5 appears to be the synthesis of a specific protease (pregnancy-associated plasma protein-A) in DF (25, 28, 29).
We have made use of the reported differences in follicular fluid profiles of IGFBP to distinguish future DF from SF at a time point preceding selection and to isolate genes differentially expressed in granulosa cells derived from these future DF. Although several studies have examined the transcription of genes of the above-mentioned candidate pathways around the time of follicular dominance establishment, no screens for genes associated with follicular selection have been reported. Using suppression subtractive hybridization, we report here on the discovery of four DF-specific genes, three of which have not hitherto been associated with this important step during folliculogenesis in monoovulatory species.
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Materials and Methods |
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Experiment 1 (material used for subtraction)
Heifer estrous cycles were synchronized as previously described (30). Briefly, heifers were fitted with an intravaginal CIDR-B device (DEC InterAg, Hamilton, New Zealand) for 10 d with an im injection of 250 µg prostaglandin F2
(PGF2; Estroplan, Parnell Laboratories, Auckland, New Zealand) 6 d after insertion of the device. Ovarian follicular development was monitored every 8 h after the PGF2 injection and continued until the time of ovary collection, using transrectal ultrasonography with a 7.5-MHz transducer (Aloka DX210, Medtel, Auckland, New Zealand). Ovary maps were drawn of the size and location of all follicles 3–4 mm or more in diameter throughout the duration of ultrasonography. On d 1.6–2.3 after ovulation, as monitored by the disappearance of the largest follicle, heifers were killed at the local abattoir, and both ovaries were removed and placed directly on ice until being transported to the laboratory.
Experiment 2 (material used for time-course analysis)
Two im injections of PGF2 were given 14 d apart to synchronize the estrous cycle. Twelve hours after the onset of estrus, all follicles 4 mm or larger were ablated by ultrasound-guided transvaginal aspiration of follicle contents, thus avoiding the inclusion of follicles from the previous follicular wave. The day of ovulation (d 0) was defined by either natural ovulation or, in cases where the largest follicle had not yet ruptured, as the time of the follicle ablation procedure. Heifers were killed at the local abattoir on d 1.5 (n = 4), d 2.5 (n = 4), and d 3.5 (n = 7) postovulation. Both ovaries were removed and placed directly on ice until follicle removal.
Collection of material in both experiments was as follows. All follicles 4 mm or larger were dissected out, and external diameters of the follicles were measured. Follicular fluid (FF) was collected using a 16-gauge needle with a 3-ml syringe and was centrifuged at 2000 x g for 3 min. The FF (supernatant) was flash-frozen in liquid nitrogen and stored at -80 C. The granulosa cell pellet was washed in cold PBS and centrifuged again at 2000 x g for 3 min, and the pellet was flash-frozen and stored at -80 C.
Western ligand blots
IGFBPs in FF were analyzed by Western ligand blotting following the method of Hossenlopp (31). Briefly, samples (2 µl) were analyzed by 12% SDS-PAGE under nonreducing conditions. All FF samples from one heifer were run on a single gel, so that no gel to gel interactions needed to be considered. After gel electrophoresis, proteins were transferred to nitrocellulose paper (Midwest Scientific, St. Louis, MO) overnight. The nitrocellulose blots were incubated with iodinated IGF-II as previously described in detail by Peterson et al. (32). The blots were exposed to x-ray film for 1–5 d at -80 C, and band intensity was determined by densitometric scanning using Molecular Analyst (Bio-Rad Laboratories, Hercules, CA).
RNA extraction
Total RNA was extracted from frozen granulosa cells using TRIzol (Invitrogen, Auckland, New Zealand) according to the manufacturer’s instructions. Poly(A)+ RNA was extracted using PolyATract mRNA Isolation System II (Promega, Auckland, New Zealand). In the first experiment, granulosa cells from putative DF (n = 6) and SF (n = 7), as defined by low or high levels, respectively, of IGFBP-2, -4, and -5, were pooled for RNA extraction. In the second experiment, granulosa cells from individual follicles were used for RNA extraction. RNA samples were treated with deoxyribonuclease (DNase) as follows: 1 µl 0.1 M dithiothreitol, 2 µl 10x DNase reaction buffer, 1 µl RNasin, and 1 µl DNase I were added to each 15 µl RNA sample and incubated for 30 min at 37 C, followed by a phenol/chloroform extraction and ethanol precipitation, and were resuspended in 11 µl diethylpyrocarbonate-treated water. One microliter of RNA was kept as the reverse transcriptase-negative control for use in real-time PCR.
First strand cDNA was generated from either pooled poly(A)+ RNA or individual total RNA samples for use as templates for real-time RT-PCR following the Superscript II First Strand cDNA synthesis protocol (Invitrogen, San Diego, CA).
Suppression subtractive hybridization (SSH)
SSH was carried out as described in the Clontech PCR-Select cDNA Subtraction Kit user manual (Clontech, Palo Alto, CA). Poly(A)+ RNA (1.5 µg) was used for synthesis of tester (DF) and driver (SF) cDNA for use in SSH. After second strand cDNA synthesis, samples were digested with RsaI to generate shorter, blunt-ended, double-stranded cDNA fragments, necessary for adaptor ligation. Only the forward subtraction was performed, which selected for genes that were enriched in the dominant compared with the subordinate population. RsaI-digested tester cDNA was ligated with either Adaptor 1 or Adaptor 2R for use in the subtraction. Adaptor-ligated tester cDNA was denatured and hybridized with excess driver cDNA. Fresh denatured driver DNA was added, and a second hybridization was performed. The samples were PCR amplified; the primary PCR was 27 cycles, and the secondary was 12 cycles. Subtraction efficiency was analyzed by PCR comparing the abundance of known genes before and after subtraction. GAPDH was used as a nondifferentially expressed housekeeping gene (primers: 3'-CTGTTGAAGTCGCAGGAGAC and 5'-TATCATCCCTGCTTCACTG) and p450 aromatase (refer to Table 2
for primer sequences) as a potentially differentially expressed gene (see introduction). PCR was performed in a DNA thermocycler (PTC 200, MJ Research, Waltham, MA) for 18, 23, 28, and 33 cycles, with each cycle consisting of 30 sec of denaturation at 94 C, 30 sec of annealing at 56 C, and 2 min of extension at 68 C.
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Differential screening and analysis of cloned SSH cDNA
The relative abundance of clones from the subtraction was estimated by dot-blot analysis. PCR products of the subtracted population were spotted onto nitrocellulose membranes in duplicate and probed with 32P-labeled probes made from either the dominant or subordinate pooled populations using the RediPrime II random prime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ). Clones that were recognized by the dominant and not the subordinate probe were considered to be differentially expressed and sequenced (University of Waikato Sequencing Facility, Hamilton, New Zealand). Sequence homology comparison was performed using the Basic Local Alignment Search Tool, BLAST (33).
Quantitative real-time PCR
Expression of the clones confirmed to be differentially expressed through differential screening was further evaluated by real-time quantitative RT-PCR using SYBR Green (34). Primers were designed using the GCG program Prime (Accelrys, Inc., San Diego, CA; Table 2
). Primers were designed to span over intron positions to avoid false results from genomic DNA amplification. Samples were run in duplicate or triplicate. Each real-time reaction (15 µl) contained 7.5 µl SYBR Green Master Mix (PE Applied Biosystems, Foster City, CA), 5.5 µl sterile deionized water, 1 µl cDNA template, and 1 µl primer pair (100 nM). The PCR was carried out in the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycling program was 95 C for 5 min, followed by 40 cycles of 95 C for 15 sec, 56 C for 30 sec, 72 C for 30 sec, and 78 C for 10 sec. ß-Actin, a housekeeping gene, was used to normalize for variations in the amount of starting material. Dissociation curve analysis was run after each real-time experiment to ensure that there was only one product and that no primer dimers were present. To control for false positives, a reverse transcriptase-negative control was run for each template and primer pair. Real-time PCR products were verified on a 2% agarose gel.
Statistical analysis
The threshold cycle number (CT) for the each gene was generated by real-time PCR and used to quantify the relative abundance of each gene. The normalized threshold cycle number, 
CT, was calculated as: CT[gene X] - CT[actin]. The abundance of a gene was calculated relative to the sample in a series of follicles showing the least abundance and assuming an optimal amplification efficiency of 2: relative abundance of gene X in sample i = 2-CT, where -CT = CT(i) - CT(i0) and i0 is the sample with the highest CT value in a series. The variation between the duplicates was pooled over animals and genes because there was no evidence of differing variabilities between these. The pooled SD was used in the t tests to determine significance levels between follicles.
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Results |
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). We observed that the lowest levels of IGFBP-2 and -4/-5 always correlated with the largest follicle size (Fig. 2). However, with the exception of heifer 102, the levels of the three follicles (per heifer) showing the lowest levels of IGFBP-2 or IGFBP-4/-5 were too similar to allow an unambiguous prediction of the future DF. We thus used size as well as IGFBP profile criteria to select granulosa cells for the dominant pool (follicle 1 of heifers 101–106 referred to in short as DF). Conversely, only follicles with substantially higher levels of IGFBP-4 and -5 (follicles 101-6, 102-4, 103-3, 105-4, and 106-6,7), but not showing signs of protein degradation (105-6 and 106-9) were used to prepare the SF material (Fig. 2).
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). Altogether, 1600 clones were picked and PCR amplified. Ninety-four percent of the clones were verified to contain a DNA insert. The insert size ranged from 100–800 bp, with most clones lying in the range of 300–500 bp.
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, with the clones selected as enriched in dominant follicles underlined. We used a stringent judgement, selecting only those spots that by inspection showed an obvious difference in signal. Thus, 25 of the 1600 clones (1.6%) were chosen (Table 1). Using this approach differentially expressed, but rare, transcripts would not have been detected, as they would have been present in numbers too low in the RNA used for the preparation of the probe to yield a signal under the conditions used. Several such clones yielding no signal in either blot are apparent in Fig. 4 and are partly responsible for the low percentage of differentially expressed genes selected.
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). Inhibin ßA, the gene coding for 1 of the subunits of activin AB and inhibin A and both subunits of the activin A dimer, was isolated 4 times. Three of these clones are located on different regions of the gene, indicating independent PCR amplification events as opposed to having reached saturation density and reisolating the same clones in the subtraction library. Whereas inhibin and activin have been suggested to be involved during selection (see introduction), the other 21 genes have not hitherto been linked to follicular development.
Comparison of the cDNA sequence of the human or mouse orthologues with the published genomic sequences revealed the presence of introns, the position of which was assumed to be conserved in cattle, thus allowing the design of primers for real-time PCR amplicons across intron positions (Table 2). Quantitative SYBR Green-based real-time RT-PCR was performed using the pooled granulosa mRNA derived from large follicles low in IGFBP-2/-4/-5 (DF) vs. the SF material. Six of the 22 genes showed an increased abundance of at least 1.3-fold in the DF population (Table 2). These were analyzed further.
Expression levels of DF-specific genes in follicles before and after selection
We obtained fresh follicular material using a slightly modified procedure (see Materials and Methods), allowing a more accurate timing of the start of the follicular wave and removing atretic follicles from the previous follicular wave. All of the largest follicles 1.5, 2.5, 3.5, and 7 d after ablation of the DF of the previous wave were measured, and the IGFBP-2 levels in the FF were determined (Table 3). The corresponding granulosa cell total RNA was then analyzed by quantitative real-time RT-PCR for expression of the six DF-enriched genes and aromatase (Table 3).
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and Fig. 5D). In contrast, equilibrative nucleoside transporter 1 (ENT1) and lysyl hydroxylase 2 transcript levels did not show any consistent correlation with follicle type at these stages. Intrafollicular levels of IGFBP-2 were always lowest in the DF (Table 3). Thus, we have isolated four novel reliable markers for granulosa cells derived from DF.
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). In contrast, the highest levels of aromatase, inhibin ßA, apoER2, and CPD transcripts converged on a single follicle on d 2.5, and on d 1.5 marked one or more of the largest follicles (Fig. 5, A–C). MAPKKK5 levels do not correlate with follicular size on d 1.5, but do correlate on d 2.5 (in two of three cases), with the follicle showing higher levels of the other DF-specific genes (Table 3).
Thus, pre- or periselection, granulosa cell transcript levels of four of the novel DF markers as well as aromatase allow the differentiation of large (>6 mm) from small (<6 mm) follicles. Furthermore, differences in gene transcript levels are becoming apparent and in some cases significant among the large follicles (inhibin ßA, d 1.5; aromatase, d 2.5), thus indicating one follicle of a cohort as being molecularly distinct and possibly becoming the future DF (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SSH technique was used here in preference to other methods because simultaneously with the subtraction it incorporates a normalization based on the suppression PCR effect. Thus, less abundant messages are enriched up to 1000-fold (35). However, a drawback of SSH can be the presence of background clones or false positives. This problem is seen particularly in high complexity cDNA samples and where the number of differences between cDNA samples is small (35). We suspect that the second condition applies to our case where, in essence, healthy granulosa cells from follicles at nearly identical developmental stages are compared. This is reflected by the low percentage (<2%) of true positives we obtained and validates the necessity of performing a differential screen on the SSH library. Furthermore, it should be pointed out that this screen was not exhaustive, as indicated by the fact that only a subset of known differentially expressed genes was found. SYBR Green-based kinetic (real-time) RT-PCR allowed accurate quantitation of relative mRNA levels, as this highly sensitive method gives very reproducible results requiring minimal amounts of starting material. However, the use of the DNA intercalator SYBR Green for fluorescent kinetic quantitation of PCR products necessitated careful analysis of the specificity of the PCR reactions by dissociation curve analysis and gel electrophoresis. Using this method, only 6 of the 22 genes selected in the differential screen remained significantly enriched in the DF. The differences ranged from a low of 1.3-fold for apoER2 to a high of 5.4-fold enrichment for lysyl hydroxylase 2. The method used has thus led to the successful identification of genes, the expression of which is up-regulated in granulosa cells of those follicles exhibiting the lowest fluid levels of IGFBP-2, -4, and -5 and which are of largest diameter, even before selection/deviation has taken place.
Considering that the pooled DF material used for the subtractive screen could not unambiguously be identified as future DF because it was collected invasively before selection/deviation had taken place, the questions immediately arise of 1) whether the identified genes are associated specifically with the DF once it can be identified as such; and 2) whether they could be used for predicting the future DF before selection. We therefore repeated the quantitative analysis at different time points relative to selection and looked at individual follicles. Furthermore, the zero time point of the follicular wave was defined accurately, as only PG-synchronized heifers with an intact DF of the previous wave were used, and the DF ablated to induce the transient rise in FSH marking the recruitment/cohort emergence process (10). The aromatase (cyp19) gene expression was also included in these studies as a gene known to be up-regulated in granulosa cells of the DF (21). The highest transcript levels of ENT1 and lysyl hydroxylase 2 are not linked to dominance beyond d 3, but are associated with the set of largest follicles earlier in folliculogenesis when looking at pooled samples (experiment 1). In contrast, inhibin ßA, apoER2, MAPKKK5, carboxypeptidase D, and aromatase are excellent DF markers 3.5 d after follicle ablation/ovulation when the DF has reached a diameter of about 12 mm. This first, now LH-dependent, DF of the cycle usually continues to grow until about d 7, whereafter the developing corpus luteum negatively regulates the LH pulse pattern with its progesterone secretion (3). All of the novel DF markers continue to be expressed at high levels on d 7, suggesting that these genes may have a functional role in DF viability. What is the transcriptional status of these genes at earlier stages? At a time point well before selection has taken place (d 1.5), relative mRNA levels of inhibin ßA, apoER2, and aromatase, but not MAPKKK5 and CPD, clearly discriminate between the largest three and the next largest follicles, suggesting that transcription of these genes is correlated with the selection process leading to molecular differentiation of the DF. Around the time point of selection/deviation (d 2.5), when the largest follicles are approximately 8 mm, transcript levels of CPD are also much greater in the largest follicles compared with the smaller subordinates. In one d 1.5 (heifer 237) and one d 2.5 set of follicles (heifer 202), mRNA levels of inhibin ßA, apoER2, and aromatase are specifically higher in a one of the large follicles, suggesting that this follicle corresponds to the future DF. In the other two heifers on d 2.5, two follicles share the highest levels, thus precluding a prediction as to which would become dominant. Such ambiguity may be explained by the observation that in about 20% of the follicular waves in heifers no clear deviation is seen morphologically as a result of double dominance (12). In light of the developmental profile of the genes analyzed, it is likely that a follicle showing precocious expression of inhibin ßA, aromatase, and apoER2 and, 1 d later, of CPD and MAPKKK5 is likely to become the DF. Whether this correlation is causal, and whether follicles exhibiting the highest levels of these markers at early stages during folliculogenesis indeed correlate to the future DF will be the subject of future studies. However, it is intriguing that all of these genes show a strong link to endocrine factors (see below).
Our results regarding the expression of aromatase complement and extend previous reports based on in situ hybridization (21, 36). We observed at least 70-fold higher levels of aromatase in the DF relative to the next largest SF postselection/deviation. Our results suggest that the time point of differential aromatase expression can precede selection, as evidenced by animal 237 (d 1.5). This would suggest that the increase in FF levels of estrogen seen in the future DF just before selection in a recent study (18) may be due to an increase in cytochrome p450 aromatase mediated at the transcriptional level.
Several studies have examined the relative FF concentrations of inhibin A and activin around the time when selection occurs (14, 17, 18, 37, 38). Both free activin and the higher molecular weight precursor forms of inhibin A increased with increasing follicular size, but no significant difference in the concentration of these factors could be detected among the three largest follicles around the time of selection (17, 18). None of these studies examined the mRNA levels of these genes, and studies in rodents are not readily applicable to monoovulatory species (39, 40). In our screen for DF-associated genes, inhibin ßA clones were picked four times, and our real-time PCR results indicated that 1) mRNA levels show a positive correlation with follicular size; 2) before selection/deviation, levels are often significantly higher in one of the three largest follicles; and 3) at selection, the largest two or three follicles exhibit similar levels, whereas 4) after selection, the DF has the highest levels of transcripts. As inhibin ßA codes for the ß-subunit of both dimeric growth factors, activin (A and AB) and inhibin A, these results are compatible with the above-mentioned studies and suggest that differential transcription/degradation of inhibin ßA in granulosa cells underlies the observed FF concentrations of activin and inhibin A. As activin has been shown to promote granulosa cell proliferation and potentiate FSH action by increasing FSH receptor expression in vitro (41), increased expression would be expected to lead to a developmental advantage.
The observation of greater abundance of apoER2 mRNA in the DF near the time of deviation is very exciting. ApoER2, also termed LPR8, is a member of the low density lipoprotein receptor family, all of which can transport macromolecules into cells by internalizing via clathrin-coated pits and moving to the endosome to discharge their cargo (42). In contrast to its name, ApoER2 does not require apolipoprotein E for binding cholesterol-rich low or very low density lipoproteins (43). Biochemical studies further suggest that low density lipoprotein/very low density lipoprotein binding as well as internalization are not the main functions of ApoER2 (44, 45). Instead, loss of function studies in mice (46) and binding assays (47) have demonstrated that ApoER2 is a receptor for the neural signaling molecule reelin. The binding of reelin results in the phosphorylation of the adaptor protein Disabled-1 bound to the cytoplasmic receptor tail, thereby activating the downstream signaling pathway. Furthermore, different splicing of the ApoER2 gene results in many variants (45, 48), some of which have been shown to interact with members of the c-Jun N-terminal kinase signaling pathway (49) or to be secreted as dominant-negative receptors (50). The role of the up-regulation of this receptor in DF could be linked to the uptake of androgen (51), which as a substrate of aromatase is required for estrogen production by granulosa cells of the largest follicles. Alternatively, ApoER2 in granulosa cells may be involved in a signal-transducing function analogous to its role in the brain. In either event, the identification of this molecule in our screen opens up a novel line of research for understanding folliculogenesis.
MAPKK kinase 5 (MAPKKK5), also known as Ask1 (apoptosis signal-regulating kinase 1), activates both the c-Jun N-terminal kinase and p38 MAPK pathways (52). It mediates and is required for apoptosis induced by oxidative and endoplasmic reticulum stress as well as by proinflammatory cytokines such as TNF (52, 53). Recently, it was shown that apoptosis mediated by MAPKKK5 can be inhibited by the type 1 IGF receptor (54). The higher RNA levels of MAPKKK5 that we observed in the largest follicles around the time of deviation would thus sensitize the granulosa cells to apoptosis, particularly under conditions of low intrafollicular free IGF-I concentrations. The DF, with its higher free interfollicular IGF-I levels (16, 18), would thus remain immune to apoptosis, whereas SF would succumb. However, recent evidence has also suggested an involvement of MAPKKK5 in differentiation and cell survival (55), and high MAPKKK5 mRNA levels initially in the largest follicles and after selection in the DF may be related to these biological roles.
CPD is a ubiquitous member of the N/E subfamily of metallocarboxypeptidases and is enriched in the trans-Golgi network (56). CPD is thought to function by following the action of endopeptidases, such as furin and proprotein convertase 7, in the processing of proteins that transit the secretory pathway (57). Likely substrates include precursors for neuroendocrine peptides, growth factors, and receptor precursors, including IGFs and bone morphogenic protein-4 (57, 58), and possibly, via furin, a splice variant of ApoER2 (50). Whether the increased levels of CPD that we observed in DF are functionally involved in one of these signaling pathways is an area of active investigation.
Follicular dominance represents an interesting model for the convergence of multiple autocrine, paracrine, and endocrine influences leading to the selective survival of one of many initially apparently identical entities. Very little is known about the underlying molecules involved in achieving this exquisite selectivity, yet such knowledge would be valuable in human artificial reproductive technologies. This work has revealed the novel association of four genes as well as aromatase with follicular dominance, thereby opening several new avenues of research into the mechanisms underlying the establishment and maintenance of dominance in monoovulatory species.
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Acknowledgments |
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Footnotes |
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Abbreviations: apoER2, Apolipoprotein E receptor 2; CPD, carboxypeptidase D; CT, threshold cycle number; DF, dominant follicle; DNase, deoxyribonuclease; ENT, equilibrative nucleoside transporter 1; FF, follicular fluid; IGFBP, IGF-binding protein; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase 5; PGF2, prostaglandin F2; SF, subordinate follicle; SSH, suppression subtractive hybridization.
Received April 17, 2003.
Accepted for publication June 9, 2003.
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