Mol. Hum. Reprod. Advance Access originally published online on February 21, 2005
Molecular Human Reproduction 2005 11(3):151-159; doi:10.1093/molehr/gah157
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Discovery of LH-regulated genes in the primate corpus luteum
1Department of Environmental & Biomolecular Systems, OGI School of Science & Engineering, 2Division of Reproductive Sciences, 3OHSU Gene Microarray Shared Resource, Oregon National Primate Research Center, Beaverton, OR 97006, USA 4Department of Physiology/Pharmacology, Oregon Health & Science University, and 5Department of Obstetrics/Gynecology, Oregon Health & Science University, Portland, OR 97239, USA
6 To whom correspondence should be addressed at: Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR 97006, USA. Email: stouffri{at}ohsu.edu
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Abstract |
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Circulating LH is essential for the development and function of the primate corpus luteum (CL) during the menstrual cycle. However, the cellular and molecular processes whereby LH controls luteal structure and function are poorly understood. Therefore, studies were initiated to identify gene products that are regulated by gonadotrophin in the monkey CL. Rhesus monkeys either were untreated (controls, CTRL; n=3) or received the GnRH antagonist Antide (ANT; 3 mg/kg body weight, n=3) to inhibit pituitary LH secretion on day 6 of the luteal phase in spontaneous menstrual cycles. The CL was removed 24 h later. RNA was extracted and converted to cDNA. The CTRL and ANT cDNA were differentially labelled with fluorescent dyes (Cy3-CTRL and Cy5-ANT) and hybridized onto microarrays containing 11 600 human cDNA. The selected cDNA were analysed further via semi-quantitative RT–PCR (a) to validate the microarray results and (b) to determine if their expression varies in the CL (n=3/stage) between the mid (day 6–8), late (day 14–16), or very late (day 18–19, menses) luteal phase of the natural cycle. After normalization of the fluorescence data, 206 cDNA (1.8% of the total) exhibited
2-fold change in expression after ANT. Of the 25 cDNA exhibiting a 6-fold change, 6 were up-regulated and 19 were down-regulated. Twenty-two of these 25 cDNA were validated by RT–PCR as differentially expressed in the ANT group, relative to the CTRL group, and 11 of 25 changed (P<0.05) correspondingly in the late-to-very late luteal phase. Thus, we have identified gene products that are regulated by gonadotrophin in the primate CL that may be important in luteal regression during the menstrual cycle. Key words: cDNA microarray/corpus luteum/LH
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Introduction |
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The development and maintenance of the functional corpus luteum (CL) in primates during the menstrual cycle requires the actions of the pituitary-derived gonadotrophin, LH (Zeleznik and Benyo, 1994
; Niswender et al., 2000; Stouffer, 2003). Several lines of investigation indicate a critical role for LH in supporting the proper structure and function of the primate CL. The inhibition of LH synthesis and secretion (by lesioning the arcuate nucleus in the hypothalamus or administering a GnRH antagonist; Hutchison and Zeleznik, 1984; Collins et al., 1986; Dubourdieu et al., 1991; Duffy et al., 1999) or action (by administering a neutralizing LH antibody; Asch et al., 1981; Groff et al., 1984) reduces progesterone production to a level that results in premature luteal regression as evidenced by menstruation. Replacement of LH in such protocols (Hutchison and Zeleznik, 1984), notably with an escalating dose-regimen (Duffy et al., 1999), leads to the maintenance of luteal function as determined by the continued progesterone production, as well as normal luteal phase length. From these studies, it is clear that LH is an essential ‘luteotropin’ required for the primate CL during the menstrual cycle. Nevertheless, the intracellular processes and mechanisms whereby LH acts to develop and maintain primate CL structure and function are not well understood.
Previous investigations identified individual LH-regulated genes in the primate CL of the menstrual cycle that are involved in steroid synthesis or action, such as steroidogenic enzymes (Ravindranath et al., 1992a), steroidogenic acute regulatory protein (STAR; Devoto et al., 2004) and estrogen receptor (ERß; Duffy et al., 2000), or tissue remodelling, e.g. members of the matrix metalloproteinase family (Young and Stouffer, 2004) and angiogenic factors (Ravindranath et al., 1992b). Differential mRNA display was also used to identify novel genes that are regulated by LH in the ovulatory, luteinizing follicle of the rat (Espey and Richards, 2002) and the CL of the non-human primate (Yadav et al., 2004). However, relatively little information exists regarding the specific genes that are under the regulatory control of gonadotrophin in the primate CL.
The present study was initiated, therefore, to systematically investigate those genes that are acutely dependent on gonadotrophin for expression in the monkey CL, following its development in the natural menstrual cycle (Xu et al., 2003b). Using spotted (human cDNA) microarrays, the mRNA of 11 600 genes were compared in CL from control and GnRH antagonist-treated monkeys. Twenty-five cDNA exhibiting a 6-fold change among groups were further analysed by semi-quantitative RT–PCR. In addition, since there is evidence for less LH support (fewer LH pulses in the circulation; Ellinwood et al., 1984) and reduced CL sensitivity to LH (Duffy et al., 1999) in the late luteal phase of the menstrual cycle, we tested the hypothesis that LH-regulated genes will display a similar change of expression during spontaneous luteal regression as during GnRH antagonist-induced luteolysis.
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Materials and methods |
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Animal treatment and hormone assays
The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC, Beaverton, OR) were described previously (Molskness et al., 1987
). Beginning 6 days after the onset of menses, daily blood samples were collected by saphenous venipuncture during the follicular and luteal phase up to the day of luteectomy. Serum was separated and assayed for hormone concentrations by a specific electrochemoluminescent assay using a Roche Elecsys 2010 analyser (Roche Diagnostics Corporation, Indianapolis, IN) in the Endocrine Services Laboratory, ONPRC (Young et al., 2003a). The first day of low (<100 pg/ml) serum estradiol following the midcycle estradiol peak typically corresponds with the day after the LH surge, and is therefore termed day 1 of the luteal phase (Duffy et al., 1999). All protocols were approved by the ONPRC Animal Care and Use Committee, and conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals.
To block pituitary LH release, the GnRH antagonist Antide (ANT; Leal et al., 1988) was administered by s.c. injection in a vehicle of 50% propylene glycol and 50% water. ANT was synthesized at the Salk Institute for Biological Studies (San Diego, CA) and made available by the Contraceptive Development Branch, Center for Population Research, NICHD (Rockville, MD). Monkeys (n=3) were injected with ANT (3 mg/kg body weight) at 0800 h on day 6 of the luteal phase. CL was isolated and dissected 24 h later (day 7 of the luteal phase) from anesthetized monkeys during an aseptic ventral midline laparotomy (Duffy et al., 2000). Control (CTRL) animals (n=3) received no ANT injection prior to CL removal on day 7. The samples were immediately flash frozen in liquid nitrogen and stored at –80 °C until processed.
RNA extraction and spotted cDNA microarray technique
RNA was extracted from each CL using TRIzol (Invitrogen Corporation, Carlsbad, CA) according to standard protocols. RNA was further purified using an RNeasy column (QIAGEN Inc., Valencia, CA) according to the manufacturer's directions. RNA (10 µg pool, formed from the three CL comprising either the CTRL or ANT group) was converted into cDNA with the appropriately labelled nucleotide mixture (biotin-11–dCTP for the CTRL cDNA and fluorescein-12–dCTP for the ANT cDNA; PerkinElmer Life and Analytical Sciences Inc., Boston, MA). The CTRL and ANT cDNA were then hybridized into two sets of DNA array slides (Human Spotted Array Set A and Set B), each corresponding to 5800 independent human Integrated Molecular Analysis of Genomes and their Expression (IMAGE) clones (The American Type Culture Collection, ATCC, Manassas, VA) spotted in duplicate. A total of 11 600 human IMAGE clones were available for analysis.
The slides were first incubated with a streptavidin–horseradish peroxidase (HRP) conjugate, followed by tyramide–Cy5. The HRP liberates the tyramide from the fluorescent dye Cy5, generating a fluorescent signal corresponding to the mRNA expression level within the CTRL CL. The slides were then washed and an inactivater of HRP (HRP Inactivation Reagent) was applied to destroy the streptavidin–HRP activity. The slides were then incubated with an anti-fluorescein antibody that is also covalently conjugated to HRP, washed, and incubated with tyramide–Cy3. The fluorescent signal generated from the liberation of the tyramide from Cy3 was subsequently determined. The fluorescent signal generated from Cy3 corresponds to the mRNA expression level in the CL of ANT-treated animals. Cy3 and Cy5 fluorescence intensity was determined using a ScanArray 4000 XL scanner. Each slide was scanned at two laser intensities to obtain the greatest possible dynamic spread. Images were stored as 16-bit TIFF files and analysed using ImaGene software (BioDiscovery Inc., El Segundo, CA). The chemicals and scanner for the microarray analysis were from PerkinElmer Life and Analytical Sciences Inc. (Boston, MA).
A generic grid was generated for each batch of slides using a Syto61 dye (Molecular Probes Inc. Eugene, OR) stained array. The generic grid was overlaid on the experimental microarray image and the location and size of the circles were adjusted by ImaGene to accommodate slide-to-slide variation. Signals derived from contaminants were removed and the intensity of each spot was calculated as the mean intensity of the pixels in the image. The background intensity surrounding each spot was averaged and subtracted from the mean fluorescent value. The data were normalized according to the method of Yang et al. (2002).
Clone sequence validation
From the microarray database, genes displaying a 6-fold difference in the mRNA levels between CTRL and ANT groups were chosen for further analysis. To confirm the sequence of the corresponding spotted cDNA, individual IMAGE clones were ordered from Invitrogen Corporation (Carlsbad, CA) and streaked onto LB/agar plates containing ampicillin (50 µg/ml). Individual clones were isolated and grown overnight in 2 ml LB broth containing ampicillin (50 µg/ml). Plasmids were isolated using the Wizard Mini-Prep Kit (Promega Biosciences Inc., San Luis Obispo, CA), quantitated by UV spectroscopy and sequenced in the ONPRC Molecular and Cell Biology Core facility using an ABI 3100 automated sequencer (Applied Biosystems, Foster City, CA). Sequence data derived from the plasmid inserts were compared against the corresponding IMAGE cDNA spotted on the array using Vector NTI Suite software (version 7.0, Invitrogen Corporation, Carlsbad, CA). In 24 out of 25 cases, the validation result matched the microarray report, except for the cDNA spots corresponding to the gene ‘tumour suppressor deleted in oral cancer-related 1’ (DOC-1R), which was listed as ‘S-100L’ on the cDNA array.
Semi-quantitative RT–PCR analysis
RT was carried out on 1 µg DNase-treated RNA using Moloney Murine Leukaemia Virus reverse transcriptase (Invitrogen Corporation, Carlsbad, CA) for 2 h at 37 °C according to the supplier's protocol. For the validation of differential mRNA expression, the sequence obtained from the individual IMAGE clone was employed to generate primers using Vector NTI software (Table I). Primers were used in a PCR to analyse cDNA generated from CTRL and ANT RNA. To serve as an internal control, a parallel PCR was performed using primers specific for the macaque cyclophilin A gene (forward primer: 5'-GCTGGACCCAACACAAATG-3'; reverse primer: 5'-TCTTCTTGCTGGTCTTGCC-3'). The PCR was terminated at different cycle numbers (every three cycles) to ensure that product generation was in the linear phase of amplification. The parameters for the PCR were as follows: initial denaturation 94 °C/1.5 min, denaturation 94 °C/30 s, annealing 56–66 °C/45 s, and extension 72 °C/1 min. The Advantage 2 polymerase, reaction buffer, and nucleotides were purchased from Clontech, BD Biosciences (Palo Alto, CA). Densitometry analysis was performed using a gel documentation system and Quantity One software (Bio-Rad Laboratories, Philadelphia, PA). The resultant PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA) and sequenced by the ONPRC Molecular and Cell Biology Core to verify their identity.
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Gene expression analysis throughout the menstrual cycle
To analyse gene expression throughout the luteal phase of the natural cycle, RNA isolated from CL collected during the early (day 3–5 post LH surge), mid (day 6–8), mid-late (day 10–11), late (day 14–16) and very late (day 18–19; menses) luteal phase was converted to cDNA (Young et al., 2002
). PCR was conducted on samples from individual CL (n=3/stage) as described above, with cyclophilin A again serving as the internal control. Cycle number was chosen from the linear portion of the amplification curve for all genes analysed. After densitometry, the data were standardized to the internal control.
Statistical analysis
Statistical evaluation of the mean differences among experimental groups was performed by 1-way analysis of variance with significance level set at 0.05 using the SigmaStat software package (SPSS Inc., Chicago, IL). To identify significant differences among groups, the Student–Newman–Keuls post hoc test was used for pairwise multiple comparisons.
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Results |
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Effect of ANT treatment on serum progesterone levels
Prior to the treatment, serum progesterone concentrations (Figure 1) increased in both CTRL and ANT-treated monkeys during the early luteal phase (day 1–4 post LH surge). ANT administration on day 6 of the luteal phase significantly (P<0.05) decreased progesterone levels within 24 h, whereas levels remained unchanged in CTRL animals. On the day of luteectomy (day 7), progesterone levels were significantly lower in ANT versus CTRL animals (1.3 ± 0.8 versus 3.0 ± 0.04 ng/ml; P<0.05).
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Microarray identification of mRNA levels in the CL of ANT-treated monkeys relative to the CTRL
A spotted array containing 11 600 individual human genes was used in this study as the longer PCR-generated DNA probes would likely cross-hybridize more effectively with Rhesus macaque cDNA than shorter oligo DNA probes (i.e. Affymetrix arrays). In preliminary validation studies (not shown), RNA was extracted from rhesus fetal fibroblasts, converted to Cy5 and Cy3 labelled cDNA, and hybridized to the spotted arrays to determine the level of cross-species hybridization. When maximal background levels were subtracted, approximately 60% of the rhesus fetal fibroblast cDNA exhibited specific hybridization to the spotted human cDNA (data not shown). This level of hybridization is comparable to our previous experience with same-species (human) hybridization of tissues.
A total of 206 mRNA were detected with 2-fold difference in the expression between CL of ANT-treated and CTRL animals (Table II). Using a cut-off of either a 4-fold increase or decrease in mRNA levels, 51 genes that were differentially expressed in the monkey CL within 24 h after ANT administration were identified. The expression of 7 of the 51 genes was up-regulated following ANT treatment. In contrast, 44 genes were down-regulated after ANT administration. Most of these genes are associated with signal transduction pathways, endocrine/paracrine regulation, cell growth/proliferation, immune response, and lipid/cholesterol/steroid metabolism (Table III).
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Semi-quantitative RT–PCR validation of microarray results
Twenty-five mRNA whose expression changed
6-fold (Table II) were analysed further by semi-quantitative RT–PCR. Twenty-two of the 25 cDNA exhibited similar changes in expression with the microarray and RT–PCR analysis of ANT relative CTRL tissues (Table IV). Only one change was opposite to the microarray results (DOC-1R) and two did not change (C4A and ORF1). One example of an up-regulated gene is corticotropin releasing hormone binding protein, CRHBP, whose mRNA expression increased 10-fold in the CL of ANT-treated animals according to the microarray results. There is a 5.5-cycle difference at the linear part of the PCR curves, which corresponds to a 45-fold difference in gene expression between the CTRL and the ANT group (Figure 2A). Cyclophilin A mRNA showed the same level of expression in these (Figure 2A) and all of the CTRL and ANT cDNA samples tested (data not shown), thereby re-affirming (Young et al., 2002) the utility of this gene as an internal standard.
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Gene expression in the monkey CL during the natural menstrual cycle
Eleven of these 25 genes that were analysed by RT–PCR changed correspondingly in the late-to-very late luteal phase during luteal regression in the natural menstrual cycle (Table IV). For example, CRHBP expression in the CL increased significantly (P<0.05) in the late luteal phase during luteolysis, and then declined in the very late luteal phase at menses. Expression increased 10-fold from the mid-late to late luteal phase (Figure 2B).
There were three predominant patterns of mRNA expression throughout the natural luteal phase for the LH-responsive genes that were up-regulated by GnRH antagonist treatment (LH withdrawal). For one pattern of expression, mRNA levels were elevated in the early luteal phase, declined at mid luteal phase and rose again at late luteal phase (Figure 3A); this pattern included the genes for LMO7 and DOC-1R. In a second pattern, mRNA levels increased progressively from early to late luteal phase, and then declined in the very late stage (Figure 3B); examples of genes displaying this pattern were PASPA1 and PIK3R1. Lastly, mRNA levels were detectable throughout, but increased abruptly in the late luteal phase (Figure 3C); a gene that displayed this pattern was CRHBP.
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There were two patterns of mRNA expression during the luteal phase for genes that were down-regulated following ANT treatment (LH withdrawal). Most of the down-regulated genes displayed mRNA levels that were elevated throughout the luteal phase, until decline in the late luteal stage (Figure 4A); examples included HSD3B1, CDO1, ALAS1, DP1L1, and SC4MOL. The others had low mRNA levels in the early luteal phase, which increased in the mid-to-late stages, and then declined by the very late luteal phase (Figure 4B); genes that displayed this pattern included SORD and LGALS7.
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Discussion |
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Recent studies established that the overall high degree of sequence similarity (
97%) between humans and Rhesus macaque genes allows for the effective use of monkey material with human arrays (Wang et al., 2004). We set out, therefore, to identify genes in the monkey CL that are under the regulatory control of LH utilizing a human spotted cDNA microarray system. To ensure that the genes identified using such a microarray technique are in fact differentially expressed, emphasis was placed on post-array validation studies that included semi-quantitative RT–PCR. To further focus our efforts, genes differentially expressed following ANT suppression of gonadotrophin secretion (6-fold change) were analysed for corresponding changes in expression during luteal regression in spontaneously occurring menstrual cycles.
After background subtraction and normalization of the array data according to the method outlined by Yang et al. (2002), 206 cDNA were identified that show a 2-fold change in expression in the monkey CL after ANT treatment. At the level of a 4-fold change in expression, 51 cDNA were differentially expressed. Those cDNA exhibiting a 4-fold change in expression after ANT treatment were subsequently classified according to the functional domains and published information. Genes encoding proteins involved in signal transduction, transcriptional regulation, cell growth and proliferation, as well as immune function were identified as differentially expressed in the monkey CL after LH withdrawal. The majority of the differentially expressed cDNA identified were down-regulated following ANT administration (86.3%), which suggests that LH serves to predominantly stimulate rather than inhibit gene expression in the monkey CL. The fewer (13.7%) cDNA up-regulated following ANT treatment are endocrine and paracrine factors or signal transduction intermediates. As such, the proteins encoded by these up-regulated genes following LH withdrawal may serve as key initiators or intermediates required for luteal regression. In contrast, those genes down-regulated following the loss of LH support may be required for promoting luteal activities.
LH is critical for luteal cell progesterone production by maintaining the expression of several genes that encode critical steroidogenic enzymes (Duncan et al., 1999; Niswender et al., 2000). For example, 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1) expression is elevated following LH addition to human granulosa cell cultures (Sasson et al., 2004). Also, HSD3B1 mRNA levels decrease during luteal regression in the monkey CL during natural menstrual cycles (Young et al., 2003b). Accordingly, our microarray data demonstrated that HSD3B1 mRNA levels decreased in the monkey CL after ANT administration. In contrast, certain well-studied LH-regulated genes encoding proteins involved in steroidogenesis that are expected to change following ANT treatment were not identified in the present microarray study. Such examples include the STAR (Christenson and Strauss, 2000; Devoto et al., 2004) and cytochrome P450 subfamily XIA (cholesterol side chain cleavage, CYP11A) genes (Gizard et al., 2002). Analysis of the list of human genes spotted on the array revealed that CYP11A was absent. STAR cDNA was spotted on the array but did not yield a hybridization signal above background. This is likely caused by a lack of hybridization between the monkey cDNA and the spotted human gene due to species-related sequence differences.
Those cDNA that changed by 6-fold or greater in the monkey CL after ANT treatment were subsequently selected as targets for additional analysis. Of the 25 cDNA observed to change by 6-fold, six were up-regulated (0.05% of total) and 19 were down-regulated (0.16% of total). Out of these 25 cDNA, 22 were validated as differentially expressed by RT–PCR analysis, suggesting efficient cross-species hybridization. Subsequent studies were performed to determine whether these 25 cDNA exhibit a corresponding change in mRNA expression during luteal regression in naturally occurring menstrual cycles. Of the 22 validated cDNA target genes, 11 changed correspondingly in the late-to-very late luteal phase, the period when luteal regression is well underway. These results suggest that, in some cases, mid luteal phase loss of LH support reflects the changes in gene expression that occurs during natural luteal regression. Several of the genes differentially regulated following ANT treatment, exhibited similar patterns of mRNA expression throughout the natural luteal phase. For example, the mRNA expression profile for HSD3B1 was similar to the mRNA expression profile observed for CDO1, ALAS1, DP1L1 and SC4MOL in the monkey CL through the course of a natural luteal phase. In this particular pattern, peak expression occurred during luteal development and optimal progesterone production (i.e. through the early and mid-late stages). When regression is well underway (late stage CL) or complete (very late stage CL), however, the expression of these genes drops dramatically. The mRNA expression profile for SORD was similar to that observed for LGALS7 mRNA. In this pattern, mRNA levels were low in the early luteal phase, peaked at the mid-to-late stages, and then declined again during luteal regression (late to very late stage).
Previous studies utilizing various differential analysis methodologies have been conducted with the goal of identifying LH regulated genes in the ovary (Espey and Richards, 2002; Sasson et al., 2004; Yadav et al., 2004). For example, Espey and Richards used differential display to identify genes that were up-regulated in rodents following an ovulatory stimulus (Espey and Richards, 2002; Espey et al., 2003). In their study, the expression of the ALAS, GST and MT1 genes increased through the periovulatory and luteal phases of stimulated estrous cycles. The orthologous monkey genes were also found to be expressed in the macaque CL by microarray analysis as LH-regulated (i.e. down-regulated following ANT treatment). Metallothioneins bind to and are transcriptionally regulated by heavy metals, but have not been previously implicated in the LH-dependent regulation of luteal function. In another study, Yadav and co-workers set out to identify genes regulated by LH in the Rhesus macaque CL using differential display (Yadav et al., 2004). Following GnRH antagonist treatment, seven genes were determined to be differentially expressed, including aldose reductase and low-density lipoprotein receptor. Both genes were spotted on the human array but did not yield a signal above background, again potentially due to cross-species hybridization failure. Lastly, using a microarray approach, Sasson et al. (2004) identified genes that were differentially expressed following LH addition to human granulosa cell cultures. Some of the LH-regulated genes identified in our studies, such as SC4MOL and CYP19A, were also found to be regulated in a similar manner in human granulosa cell cultures by LH.
Several genes not previously associated with LH-dependent regulation of CL function were also identified in the present study. For example, corticotropin-releasing hormone binding protein (CRHBP) mRNA expression was up-regulated after ANT treatment. The expression of CRHBP mRNA also increased significantly at the late stage, relative to all other stages of a natural luteal phase. Thus, LH may suppress the expression of CRHBP mRNA when the CL is at its peak with regard to progesterone production. The suppression of CRHBP may be lost, however, when the CL begins to undergo regression. CRH was discovered originally in the hypothalamus–pituitary axis (Vale et al., 1981), and later in the placenta (Shibasaki et al., 1982; Ni et al., 2004). It is now known that the CRH system is composed of four ligands (corticotropin, urocortin, urocortin II, and urocortin III), one binding protein (CRHBP), and two receptors (CRHR1 and CRHR2). CRHBP is known to bind CRH and urocortin, thus preventing activation of their receptors (Dautzenberg and Hauger, 2002). Some of the CRH system components, including CRH, CRHR1 and CRHBP, are also expressed in the human ovary (Asakura et al., 1997). Recently, CRH, urocortin, CRHR1 and CRHR2
have been observed specifically within the human CL (Muramatsu et al., 2001). Since the cellular and molecular mechanisms of luteolysis in primates remain obscure, the CRH system may be a key component in the initiation or execution of CL regression.
Thus, we have identified a number of genes under the regulatory control of LH that have not previously been implicated in the primate luteal function. Genes stimulated by LH, and thus suppressed following LH removal, may serve to maintain CL structure and function. Genes inhibited by LH, and thus increased in expression following LH removal, may play a role in luteal regression. Studies are now underway to evaluate the role such novel systems play in LH-mediated maintenance of luteal function and regression. Specifically, the expression of all the CRH ligands and receptors are being evaluated in the primate CL (Xu et al., 2003a). An added level of complexity in the CRH system stems from the observations that each receptor type exists as multiple protein forms due to the generation of alternatively spliced mRNA (Pisarchik and Slominski, 2001; Catalano et al., 2003; Johnson et al., 2003). Studies are also underway, therefore, to determine which receptor isoforms are expressed, and if so, whether they are regulated by LH.
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Acknowledgements |
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We are grateful to members of the Division of Animal Resources, Endocrine Services Laboratory, Molecular and Cell Biology Core Laboratory, and Spotted Microarray Core of the OHSU Gene Microarray Shared Resource for their technical assistance. This work was supported by NIH NICHD HD20869 (R.L.S.), NIH NICHD HD42000 (J.D.H.), through a cooperative agreement (U54 HD18185) as part of the Specialized Cooperative Centers Program in Reproduction Research, and NCRR RR00163 (R.L.S. and J.D.H.).
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