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Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering (J.X., R.L.S.), and Division of Reproductive Sciences, Oregon National Primate Research Center (J.X., J.D.H., R.L.S.), Beaverton, Oregon 97006; and Departments of Obstetrics/Gynecology and Physiology/Pharmacology, Oregon Health and Science University (J.D.H., R.L.S.), Portland, Oregon 97239
Address all correspondence and requests for reprints to: Dr. Richard L. Stouffer, Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: stouffri{at}ohsu.edu.
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Abstract |
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Objective: Our objective was to determine whether other components of the CRH/urocortin-receptor-binding protein (UCN-R-BP) system are expressed in the CL during the menstrual cycle and regulated by LH.
Design: CL were collected from monkeys during the early (d 3–5 after the LH surge) to very late (d 18–19) luteal phase and from controls or animals receiving GnRH antagonist (Antide, 3 mg/kg body weight). CRH/UCN-R-BP system components were quantitated for mRNA levels by real-time PCR and analyzed for protein localization by immunohistochemistry.
Results: All genes encoding the CRH/UCN-R-BP system, except for UCN3, were expressed in the CL. CRH mRNA levels did not change during the luteal phase, whereas expression of UCN, UCN2, CRHR1, and CRHR2 was maximal at early or mid luteal phase before declining (P < 0.05) at the later stages. CRHBP mRNA levels were lowest at mid and increased (P < 0.05) in the late luteal phase. Suppressing gonadotropin secretion reduced UCN2 (P < 0.05) and increased CRHBP (P < 0.05) mRNA levels, without altering the expression of other ligands or receptors. CRH, UCN, UCN2 and their receptors were localized to the granulosa-lutein cells of the CL, whereas CRHBP was limited to the theca and theca-lutein cells of the preovulatory follicle and CL.
Conclusions: A local CRH/UCN-R-BP system exists in the macaque CL that is dynamically expressed and LH regulated during the luteal phase of the menstrual cycle. Ligand-receptor activity may regulate luteal structure-function, at this point in an unknown manner, in primates.
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Introduction |
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, ß, c, d, e, f, g, h, and v_1) produced by differential splicing of exons 3–6 and 10–13. The CRHR2 gene expresses three known subtypes (, ß, and 
) that are produced by the use of alternate 5' exons (10, 11, 12). Some of the CRH/UCN-R-BP system components, including CRH, UCN, CRHR1, CRHR2, and CRHBP, were recently detected in the human ovary (13) or CL (14). However, detailed studies on the dynamics of their expression during the ovarian cycle, or their regulation by luteotropic hormones, have not been reported in any species. Therefore, the present study was initiated to test the hypotheses that 1) various components of the CRH/UCN-R-BP system were expressed in the macaque CL during the menstrual cycle, and 2) their expression was regulated by LH. The mRNAs for the CRH/UCN-R-BP components were detected initially using RT-PCR. Subsequently, real-time PCR was performed to quantitate mRNA levels in the CL at each stage of the luteal phase and after acute LH deprivation. Finally, CRH/UCN-R-BP proteins were localized to specific cell types within the primate ovary and CL by immunohistochemistry (IHC).
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Materials and Methods |
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The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC) were described previously (15). All protocols were approved by the ONPRC Animal Care and Use Committee and conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Beginning 6 d after onset of menses, daily blood samples were collected by saphenous venipuncture during the follicular and luteal phase up to the day of lutectomy. Serum was assayed for estradiol and progesterone (P) concentrations by specific electrochemoluminescent assays, as previously described (16). The first day of low (<100 pg/ml) serum estradiol after the midcycle estradiol peak typically corresponds with the day after the LH surge and is therefore termed d 1 of the luteal phase (17).
Tissue collection
To analyze luteal tissues throughout the luteal phase of the natural cycle, CL (n = 5 per stage) were collected during the early (ECL, d 3–5 after the LH surge), mid (MCL, d 6–8), mid-late (MLCL, d 10–12), late (LCL, d 14–16), and very late (VLCL, d 17–18; menses) luteal phase. These intervals provided tissues representing the developing, developed functional, on the verge of regressing, regressing, and regressed CL, respectively (18).
To analyze gene expression in the CL after acute LH withdrawal, the GnRH antagonist Antide (ANT) was administered by sc injection in a vehicle of 50% propylene glycol and 50% water, as previously described (4). Monkeys (n = 3) were injected with ANT (3 mg/kg body weight) at 0800 h on d 6 of the luteal phase. CL were isolated 24 h later (d 7 of the luteal phase) from anesthetized monkeys during an aseptic ventral midline laparotomy (19). Control (CTRL) animals (n = 3) received no ANT injection before CL removal on d 7. ANT administration on d 6 of the luteal phase significantly (P < 0.05) decreased LH (17) and P (4) levels within 24 h, whereas levels remained unchanged in CTRL animals. CL samples collected previously from CTRL and ANT-treated monkeys to evaluate LH-regulated genes (4) were used in the study.
To localize protein expression in the individual cell types, ovaries (n = 3 per stage) were surgically removed during the preovulatory follicular phase (d 0–1 before the LH surge) and the early to late luteal phases (stages described previously in this section) of the natural menstrual cycle. The tissue was processed for immunohistochemical analysis of proteins as described previously (20).
RNA extraction and RT-PCR analysis
RNA was extracted from each CL using TRIzol Reagent (Invitrogen Corp., Carlsbad, CA) according to standard protocols. RT was performed using 1 µg DNase-treated RNA and Moloney murine leukemia virus reverse transcriptase for 1 h at 37 C according to the supplier’s protocol. The individual components of the rhesus macaque CRH/UCN-R-BP system (CRH, UCN, UCN2, UCN3, CRHR1, CRHR2, and CRHBP) were PCR amplified with specific primers generated from human sequences (www.ncbi.nlm.nih.gov) (Table 1
). 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 parameters for the PCR were as follows: initial denaturation 94 C for 1.5 min, denaturation at 94 C for 30 sec, annealing for 45 sec, and extension at 72 C for 1 min. All PCR reagents were purchased from BD Biosciences (San Jose, CA). The resultant PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN Inc., Valencia, CA) and sequenced at the ONPRC Molecular and Cell Biology Core facility. Sequence data were compared against the corresponding human genes using the Vector NTI Suite software (Invitrogen).
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The partial sequences of the rhesus macaque CRH/UCN-R-BP system genes were subsequently used to design real-time PCR primer and TaqMan probe sets using Primer Express software from Applied Biosystems (Foster City, CA) as reported previously (18) (Table 1
). Primers were synthesized by Invitrogen, and TaqMan probes were synthesized by Applied Biosystems. Probes were labeled with the 5' reporter dye 6FAM and the 3' quencher dye MGBNFQ. A matrix of varying primer concentrations was employed to determine optimal concentrations of assay components. TaqMan PCR Core Reagents Kit and the Applied Biosystems 7900HT Fast Real-Time PCR System, with 18S rRNA serving as an internal control in each well, were used as previously described (18). The PCR were conducted with thermal cycler conditions of 2 min at 50 C, 10 min at 95 C, and 45 cycles of 15 sec at 95 C (denaturation) and 1 min at 60 C (primer annealing/extension). The number of amplification cycles for the fluorescence to reach a determined threshold level (CT) was recorded for every unknown sample and an internal standard curve. The internal standard curve, used for relative mRNA quantification, was generated from five 10-fold dilutions of pooled CL samples. The data were analyzed according to the method of Young et al. (18).
IHC
Ovaries fixed overnight in 10% neutral buffered formalin were dehydrated in 70% ethanol solutions and paraffin embedded. The 5-µm sections prepared in the Imaging and Morphology Core at ONPRC were deparaffinized and hydrated through CitriSolv Clearing Agent (Fisher Scientific, Pittsburgh, PA) and a graded series of ethanol. Sections were rehydrated in PBS, followed by incubation in 3% hydrogen peroxide/60% methanol to quench any endogenous peroxidase activity. The sections were incubated for 1 h at room temperature with the primary antihuman antibodies (1:200 for CRH, sc-1759 and UCN, sc-1825; 1:100 for CRHR1, sc-12381 and CRHR2, sc-20550; 1:400 for CRHBP, sc-1822 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; and 1:400 for UCN2, H-019–24 from Phoenix Pharmaceuticals, Inc., Belmont, CA). As negative controls, antibodies preabsorbed with blocking peptides (Santa Cruz Biotechnology and Phoenix Pharmaceuticals) at 4 C overnight were incubated on adjacent tissue sections. The slides were then incubated with the appropriate secondary antibodies and processed using an ECTASTAIN Elite ABC Kit from Vector Laboratories, Inc. (Burlingame, CA). The antigen-antibody complex was visualized by incubation with 3,3'-diaminobenzidine. The tissue was counterstained using hematoxylin and viewed via Zeiss Axioplan microscopy. A CoolSNAP CCD Camera (Photometrics Inc., Tucson, AZ) was used for image capture.
Statistical analysis
Statistical evaluation of mean differences among stages of the luteal phase was performed by one-way ANOVA with a significance level set at 0.05 using the SigmaStat software package (SPSS Inc., Chicago, IL). To identify significant differences between stages, the Student-Newman-Keuls post hoc test was used for pairwise multiple comparisons. A Student’s t test was used to compare parameters between CTRL and ANT-treated animals.
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RT-PCR analysis demonstrated that the genes encoding CRH, UCN, and UCN2, but not UCN3 (which was identified in monkey testis), were expressed in the macaque CL (Fig. 1; summarized in Table 2). Likewise, RT-PCR products for the and c isoforms of CRHR1, the CRHR2 isoform, as well as CRHBP were identified in macaque CL. CRHR1e, 1f, and CRHR2ß were not detected in the CL but were detected in positive control monkey tissues (e.g. testis and heart) (Table 2). The partial sequences of the gene products for the macaque CRH/UCN-R-BP system (data not shown), obtained by RT-PCR, have a high degree of similarity (95–98%) to the corresponding sequences from human genes.
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). However, UCN gene expression levels increased 4-fold (P < 0.05) from ECL to peak at MCL before declining significantly (P < 0.05) at the later stages (Fig. 2B). UCN2 mRNA levels were detectable throughout but decreased (P < 0.05) in the LCL compared with MCL and rose (P < 0.05) again in the VLCL (Fig. 2C). CRHR1 mRNA levels were highest in the ECL and MCL relative to the remaining stages of the luteal phase (Fig. 2D). CRHR2 mRNA increased (P < 0.05) in the MCL relative to its expression level in any of the other stages analyzed (Fig. 2E). In contrast, CRHBP mRNA levels were low at MCL and then increased 10-fold (P < 0.05) from MLCL to LCL (Fig. 2F).
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Real-time PCR analysis determined that UCN2 mRNA levels in CL decreased 2.4-fold (P < 0.05) after ANT treatment, whereas those for CRH and UCN (P = 0.13) did not significantly change. There were no significant changes in mRNA levels for either CRHR1 or CRHR2 (P = 0.09) after LH withdrawal. In contrast, CRHBP mRNA levels increased 5.1-fold (P < 0.05) after ANT treatment relative to CTRL (Table 3).
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In the late follicular phase, there was minimal specific IHC staining (compared with negative controls; not shown) for CRH or UCN in the preovulatory follicle (Fig. 3, A and B), atretic antral follicles, or smaller preantral follicles (Fig. 3A). However, specific staining for both CRH and UCN was observed in the CL (Figs. 3C and 4, A–C). The staining appeared to concentrate in granulosa-lutein cells of the CL as well as in some of the interstitial cells in the ovarian stroma (Fig. 3C), whereas theca-lutein and endothelial cells in the CL did not label (Fig. 4A). In CL removed from the different stages of the luteal phase, intense staining for both ligands were most evident in the ECL (Fig. 4A) before decreasing in the MCL (CRH; Fig. 4B) and MLCL (UCN; Fig. 4C). In contrast, appreciable staining for UCN2 was observed in the granulosa, but not theca, cells of the preovulatory follicle (Fig. 3B). UCN2 immunostaining was also evident in the granulosa cells of smaller antral and growing preantral follicles (Fig. 3, A and C). Specific immunostaining for UCN2 was particularly evident in the CL (Fig. 4, A–C). In the ECL (Fig. 4A), both steroidogenic and endothelial cells displayed strong UCN2 immunolabeling, which subsequently declined in the MLCL (Fig. 4C). No significant staining was evident in control sections processed with preabsorbed primary antibody (Fig. 4A, bottom left; representative ECL section shown).
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, A and B). In contrast, specific staining was evident in CL isolated from the different stages of the luteal phase (Figs. 5C and 6, A–C). Expression of CRHR1 was detected at high levels in granulosa-lutein cells of the CL as well as interstitial cells in the ovarian stroma but not noted in theca-lutein or endothelial cells of ECL tissue (Figs. 5C and 6A). CRHR2 staining was also present but at modest levels. However, luteal cell CRHR staining was diminished in the MCL (CRHR1; Fig. 6B) and LCL (CRHR2; Fig. 6C). Immunohistochemical staining for CRHBP was not evident in preantral follicles but was detectable only in the theca cells of preovulatory (Fig. 5B) and atretic follicles (Fig. 5A). CRHBP immunostaining was also observed in the interstitial tissue and CL during the luteal phase (Figs. 5C and 6, A–C). The theca-lutein but not the granulosa-lutein or endothelial cells of the CL were immunolabeled (Fig. 6, A and C). Although positively stained cells were detectable early in the CL lifespan (Fig. 6A), few cells were positive in the MCL for CRHBP expression (Fig. 6B). The most intense level of CRHBP staining occurred in CL obtained from the late luteal phase (Fig. 6C). No receptor or binding protein staining was evident in control sections processed with preabsorbed primary antibody (bottom left of Fig. 6A; representative ECL section shown).
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Discussion |
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The mRNA expression of all ligands, except UCN3, was detected in the rhesus macaque CL. CRH is abundantly distributed in the central nervous system (27) as well as in certain peripheral tissues, such as adrenal gland, testis, and placenta (9). UCN expression is limited to specific regions of the brain (28) but is also found in a number of organ systems that include the pituitary, thymus, and testis (29). UCN2, highly expressed in brain, is also present in adrenal gland, heart, and peripheral blood cells (23). Our evidence that CRH and UCN are also expressed in the macaque ovary is consistent with previous reports describing their presence in the human ovary (13, 14). The present study also provides the first evidence that UCN2, but not UCN3, exists in the primate CL.
The mRNA expression of both receptors and the binding protein was also detected in the rhesus macaque ovary. CRHR1 expression was previously documented in the brain and pituitary (30) with low levels also being reported in the testis and ovary (31, 32, 13, 14). The present study identified two isoforms, and c, for CRHR1 in the monkey CL. The identification of these two isoforms in the ovary may be of significance, because the amino acid differences between the various CRHR1 isoforms alters binding affinity for CRH (33). CRHR2 was the only type 2 receptor isoform identified in the monkey ovary, which is consistent with a previous analysis of CRHR expression in the human ovary (14). CRHR2 mRNA was undetectable in rat ovary (32), however, suggesting that CRHR2 gene subtype expression is species specific (10, 34). Previously, CRHR2 was mainly localized to the subcortical structures of the brain (35) but also detected in muscle (34), including myometrium (36). Other receptor variants were undetectable in the ovary, although some, such as CRHR1e, 1f, and CRHR2ß, were identified in other macaque tissues considered putative positive controls based on human studies (12, 34). Previous analysis of tissue distribution indicated that CRHBP is expressed in human liver (37), placenta (38), and brain (39). Asakura et al. (13) could not detect CRHBP mRNA expression in the human follicle and proposed that CRHBP protein in the ovary originated from the circulation. However, based on our current findings and published studies (4), CRHBP is also expressed in the macaque antral follicle and CL.
Many components of the CRH/UCN-R-BP system are dynamically expressed in the macaque CL during the luteal lifespan in the menstrual cycle. Although CRH mRNA levels did not change during the luteal phase, UCN, CRHR1, and CRHR2 expression peaked during either CL development or optimal P production (i.e. through the early and mid stages). When CL regression was underway or complete (i.e. late to very late luteal phase), however, the expression of these genes dropped dramatically. In contrast, the real-time PCR data confirmed our previous semiquantitative results (4) that CRHBP mRNA expression increased significantly in the CL during luteal regression. Our findings differ from those of Muramatsu et al. (14), who reported that CRH, UCN, and CRHR gene expression was higher during the regression of the human CL than at earlier stages of the luteal phase. This discrepancy may result from the differences between species, methods for classifying stages of the luteal phase, and/or techniques employed (e.g. RT-PCR vs. quantitative real-time PCR).
Our present study indicates that luteal UCN2 mRNA expression is up-regulated and CRHBP was down-regulated by LH, whereas other ligand and CRHR mRNAs appeared to be mostly unaffected by this luteotropic hormone. Because there is evidence for less LH support (i.e. reduced LH pulsatility) (40) and reduced CL sensitivity to LH (17) in the late luteal phase of the menstrual cycle, it is noteworthy that UCN2 and CRHBP displayed similar changes in mRNA expression during spontaneous luteal regression as during GnRH antagonist-induced LH withdrawal. Thus, LH may promote the expression of UCN2 and suppress CRHBP when the CL is at its peak function with regard to P production. This regulation may be lost, however, when the CL begins to undergo regression. The factors regulating the changes in mRNA expression for other ligands and CRHR during the natural luteal phase remain unknown, although a role for LH cannot be ruled out. For genes exhibiting a tendency to change after LH withdrawal, such as UCN (P = 0.13) and CRHR2 (P = 0.09), increasing the sample size or altering the time interval of collection may yield significant changes in mRNA levels.
This is the first report detailing the expression and localization of CRH/UCN-R-BP proteins in the ovary during the preovulatory stage of the ovarian cycle. There was no obvious immunoreactivity for CRH, UCN, and CRHR in the macaque ovary during the preovulatory phase. However, UCN2 was detected in the granulosa cells of the preantral, preovulatory, and atretic follicles as well as the interstitial cells in the ovarian stroma. In contrast, intense CRHBP staining was exclusively localized to the theca cells of the preovulatory and atretic antral follicles. Previous studies focused on selected components of the CRH/UCN-R-BP system in developing follicles. For example, CRH was reported to localize in theca cells of growing antral (7–8 mm in diameter) follicles (13) and the dominant follicle (14) in the human ovary during the follicular phase. Weak immunoreactive UCN and CRHR were detected in granulosa and theca interna cells in dominant follicles (14). CRHBP signal was in theca cells of growing antral follicles in the human ovary (13), which is consistent with our observation.
IHC results suggest that ligands (CRH, UCN, and UCN2) and receptor proteins are associated with the granulosa-lutein cells in the CL and interstitial cells in the ovarian stroma during the luteal phase. A high level of staining intensity for ligands and receptors was observed in luteal cells in the early stage of the luteal phase, suggesting that processes causing (e.g. the mid-cycle LH surge) or associated with (e.g. LH-induced local factors) ovulation or luteinization of the follicle wall influence CRH/UCN-R expression. Subsequently, ligand and receptor staining declined, whereas binding protein staining increased in the CL during the later stages of the luteal phase. The results suggest that steroidogenic cells in the CL are the sites of CRH/UCN ligand synthesis and receptor-mediated action. In contrast, CRHBP localizes to the theca-lutein and interstitial cells particularly during the late luteal phase. Previous IHC studies demonstrated that CRH, UCN, and CRHR were present in luteinized granulosa and theca cells (41, 14) and that significant CRH immunostaining was observed in developing CL and less prominent or totally absent in regressing CL of rodents and women (42, 41). These findings are consistent with our observations. However, as noted earlier, Muramatsu et al. (14) reported higher levels of immunoreactive CRH and UCN mRNA, as well as protein, in the regressing CL relative to the developing CL in women.
In summary, this study provides the first detailed analysis of the CRH/UCN-R-BP system expression in the primate ovary during the menstrual cycle. According to the mRNA data, there is a dynamic regulation of UCN, CRHR, or CRHBP gene expression in the CL during the menstrual cycle. The pattern of CRH/UCN-R-BP system mRNA expression during the natural luteal phase suggests that ligands regulate cellular processes in the CL primarily during the early luteal phase when there is greater expression of ligands/receptors and less expression of binding protein. In the late luteal phase, however, their associated activities may be restricted as the expression of the ligand/receptor decreases and the binding protein increases. This is consistent with the hypothesis that ligand-receptor action promotes luteal structure-function, and its loss is associated with luteal regression. Nevertheless, one group (43) reports that CRH suppressed estrogen and IGF-I production by human and rat granulosa cells in vitro, and as such, opposite antigonadotropic actions cannot be ruled out. Additional studies are warranted to evaluate the endocrine (LH) and local control, plus the functional significance of the CRH/UCN-R-BP system in the preovulatory follicle as well as in the maintenance/regression of the CL. Because the cellular and molecular mechanisms of luteolysis in primates remain obscure, the CRH/UCN-R-BP system may represent a previously unappreciated component in the initiation or execution of CL regression.
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Acknowledgments |
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Footnotes |
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The authors J.X., J.D.H., and R.L.S. have no potential conflicts of interest in this research.
First Published Online January 31, 2006
Abbreviations: ANT, Antide; CL, corpus luteum; CRHBP, CRH-binding protein; ECL, early CL; IHC, immunohistochemistry; LCL, late CL; MCL, mid CL; MLCL, mid-late CL; P, progesterone; UCN, urocortin; UCN-R-BP, UCN-receptor-binding protein; VLCL, very late CL.
Received December 20, 2005.
Accepted January 25, 2006.
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and ß in the rhesus monkey corpus luteum during the menstrual cycle: regulation by luteinizing hormone and progesterone. Endocrinology 141:1711–1717 receptor. Mol Endocrinol 12:1077–1085 and CRF2ß receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. Endocrinology 136:4139–4142[Abstract]This article has been cited by other articles:
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  R. L. Bogan, M. J. Murphy, R. L. Stouffer, and J. D. Hennebold Systematic Determination of Differential Gene Expression in the Primate Corpus Luteum during the Luteal Phase of the Menstrual Cycle Mol. Endocrinol., May 1, 2008; 22(5): 1260 - 1273. [Abstract] [Full Text] [PDF]
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E. Xiao, L. Xia-Zhang, N. Vulliemoz, J. Rivier, and M. Ferin Astressin B, a Corticotropin-Releasing Hormone Receptor Antagonist, Accelerates the Return to Normal Luteal Function after an Inflammatory-Like Stress Challenge in the Rhesus Monkey Endocrinology, February 1, 2007; 148(2): 841 - 848. [Abstract] [Full Text] [PDF]
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