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Endocrinology, doi:10.1210/en.2007-0541
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Endocrinology Vol. 148, No. 11 5385-5395
Copyright © 2007 by The Endocrine Society

Expression and Role of the Corticotropin-Releasing Hormone/Urocortin-Receptor-Binding Protein System in the Primate Corpus Luteum during the Menstrual Cycle

Jing Xu, Fuhua Xu, Jon D. Hennebold, Theodore A. Molskness and Richard L. Stouffer

Divisions of Neuroscience (J.X.) and Reproductive Sciences (F.X., J.D.H., T.A.M., R.L.S.), Oregon National Primate Research Center, Beaverton, Oregon 97006; and Departments of Obstetrics and Gynecology (J.D.H., R.L.S.) and Physiology and Pharmacology (R.L.S.), Oregon Health & Science University, Portland, Oregon 97239

Address all correspondence and requests for reprints to: Richard L. Stouffer, Ph.D., Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 Northwest 185th Avenue, Beaverton, Oregon 97006. E-mail: stouffri{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH/urocortin-receptor-binding protein (CRH/UCN-R-BP) mRNAs are dynamically expressed in the primate ovary during the menstrual cycle. Therefore, studies were designed to localize CRH/UCN-R-BP mRNAs to ovarian cell types, quantitate protein expression during the corpus luteum (CL) lifespan, and investigate the role of this system in the macaque ovary at midcycle. Monkey ovaries were removed during the preovulatory phase and through the luteal phase to localize CRH/UCN-R-BP mRNAs by in situ hybridization and determine their protein levels in CL by Western blotting. Also, vehicle or a CRH receptor antagonist (astressin) was injected into the preovulatory follicle; daily serum samples were analyzed for hormone levels, and ovaries were removed on d 9 of the luteal phase for histological analysis. There was minimal ligand mRNA staining, whereas receptor and CRHBP was detected in the granulosa and theca cells of the preovulatory follicle. However, ligand and receptor mRNA staining was appreciable in luteal cells of the CL during the early luteal phase (ECL) and diminished in the late luteal phase (LCL). CRHBP staining was low in the ECL and increased markedly in the LCL. Ligand and receptor protein expression was also highest during ECL, whereas CRHBP expression was highest at the LCL. Although astressin injection did not prevent follicle rupture, progesterone levels were significantly less by the mid-luteal phase, and estradiol levels never increased above baseline during the CL lifespan. Histological indices of cell degeneration were observed in the astressin-treated CL. Thus, CRH/UCN-R-BP components are expressed in an ovarian cell-specific manner. The expression pattern and results from antagonist injection are consistent with the hypothesis that CRH/UCN-R activation promotes luteal development and/or structure-function in monkeys during the menstrual cycle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEPTIDES OF THE CRH/urocortin-receptor-binding protein (CRH/UCN-R-BP) system are expressed by brain neurons and pituitary corticotropes and play a key role in neurotransmission and neuroendocrine regulation of the stress response (1). However, recent evidence suggests that the CRH/UCN-R-BP system is also present in a variety of other tissues in mammals that include the immune, cardiovascular, digestive, and reproductive systems, skin, and some types of human tumors (2, 3). The ubiquitous distribution of CRH/UCN-R-BP peptides, capable of initiating diverse signaling mechanisms in different tissues, gives this system enormous versatility and plasticity. It is currently known that the primate CRH/UCN-R-BP system is composed of four ligands (CRH, UCN, UCN2, and UCN3) and two seven-transmembrane G protein-coupled receptors (CRHR1 and CRHR2). The interactions of ligands with their receptors are modulated by a binding protein, CRHBP, with affinity for CRH and CRH-like peptides (2, 4).

Recent studies indicated that a local CRH/UCN-R-BP system exists in the primate ovary, including human and rhesus monkey (5, 6, 7, 8). The mRNAs for the CRH/UCN-R-BP components are dynamically expressed in the corpus luteum (CL) during the menstrual cycle, with UCN and CRHBP expression up-regulated and down-regulated, respectively, by the primary luteotropin, LH (8). CRH/UCN-R mRNA levels peaked in the macaque CL during either luteal development or optimal progesterone (P) production (i.e. through the early and mid stages of the luteal phase). When CL regression was underway or complete (i.e. late to very late luteal phase), the expression of these components dropped, whereas CRHBP mRNA levels increased significantly (8).

However, detailed studies designed to localize CRH/UCN-R-BP mRNA expression to specific ovarian cell types, analyze the dynamics of protein expression during the mestrual cycle, and evaluate the role(s) of the CRH/UCN-R-BP system in ovary in vivo have not been reported in any species. Therefore, in the present study, 1) CRH/UCN-R-BP mRNAs were localized to specific cell types within the macaque ovary by in situ hybridization (ISH), 2) Western blotting was performed to semiquantitate protein levels in the macaque CL at each stage of the luteal phase during the menstrual cycle, and 3) in vivo protocols were performed to test the hypothesis that a CRHR antagonist, astressin, which blocks both CRHR1 and CRHR2, would disrupt luteal development and/or function when delivered directly into the periovulatory, luteinizing follicle of the ovary during the spontaneous menstrual cycle in the rhesus monkey.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatment, hormone assays, and ovarian tissue collection
The general care and housing of rhesus monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC, Beaverton, OR) were described previously (9). Adult female monkeys exhibiting normal menstrual cycles of approximately 28 d were bled daily by saphenous venipuncture beginning 6 d after the onset of menses. Serum was separated and assayed for estradiol (E) and P concentrations by a specific electrochemoluminescent assay using a Roche Elecsys 2010 analyzer (Roche Diagnostics Corp., Indianapolis, IN) in the Endocrine Services Laboratory, ONPRC (10). The interassay variations were 6.1% for E and 5.4% for P, and the limits of sensitivity were 5 pg/ml for E and 0.03 ng/ml for P. The first day of low (<100 pg/ml) serum E after the midcycle E peak (>200 pg/ml) typically corresponds with the day after the LH surge and is therefore termed d 1 of the luteal phase (11). 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.

CL (n = 4 per stage) were collected from the early (ECL, d 3–5 after LH surge), mid (MCL, d 6–8), midlate (MLCL, d 10–12), late (LCL, d 14–16), and very late (VLCL, d 17–18; menses) luteal phase of the natural menstrual cycle and frozen in liquid nitrogen for extraction of total protein using tissue protein extraction reagent (T-PER; Pierce Biotechnology, Inc., Rockford, IL). These intervals provided tissues representing the developing, developed functional, on the verge of regression, regressing, and regressed CL, respectively (12). In addition, ovaries (n = 3 per stage) were surgically removed from rhesus monkeys during the preovulatory follicular phase (d 0–1 before LH surge) and the early to late luteal phase, fixed overnight in 10% neutral buffered formalin, dehydrated in 70% ethanol solutions, and paraffin embedded. The 5-µm serial sections were prepared in the Specialized Cooperative Centers Program in Reproduction and Infertility Research’s (U54) Imaging and Morphology Core at the ONPRC using an American Optical (Southbridge, MA) microtome and mounted on Superfrost/Plus slides (Fisher, Santa Clara, CA) (13).

ISH
To identify CRH/UCN-R-BP mRNA in the individual ovarian cell types, ISH was performed using a digoxigenin (DIG)-based riboprobe kit according to the manufacturer’s directions (Roche Applied Science, Indianapolis, IN). Briefly, primers for PCR amplification of CRH/UCN-R-BP cDNAs were synthesized by Invitrogen Corp. (Carlsbad, CA) (see Table 1Go for sequences). PCR products for ISH probe synthesis were separately subcloned into pGEM-T Easy Vector (Promega Corp., Madison, WI). DIG-labeled sense riboprobes (negative control) and complementary antisense riboprobes were generated from 1 µg linearized plasmid DNA templates in a 20-µl reaction volume containing 1x transcription buffer, 1x DIG RNA labeling mix, 20 U RNase inhibitor, and 40 U T7 or SP6 RNA polymerase (Roche Applied Science). After 2 h at 37 C, the DNA template was removed by treatment with 10 U DNase I (Promega) for 10 min at 37 C. RNA was precipitated with 1/10 vol of 5 M LiCl, and 2.5 vol of 100% ethanol at –20 C overnight, centrifuged at 16,000 x g for 15 min, washed with 100 µl 70% ethanol, centrifuged at 16,000 x g for 5 min, dried in a vacuum, and resuspended in 100 µl diethylpolycarbonate-treated water. Probe concentrations were determined empirically in side-by-side comparisons with a RNA standard (Roche Applied Science) on a 1% agarose gel.


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  		TABLE 1. Primer sequences and annealing temperature used for PCR amplification

 
Ovarian sections were deparaffinized, hydrated through CitriSolv clearing agent (Fisher Scientific, Pittsburgh, PA) and a graded series of ethanol, washed in PBS before and after postfixation with 4% paraformaldehyde/PBS for 5 min, dehydrated with a graded series of ethanol, and dried in air for 10 min. Sections were then incubated 2–4 h with prehybridization buffer, 2x sodium chloride/sodium citrate solution (SSC) containing 50% (vol/vol) deionized formamide, at room temperature. DIG-labeled RNA probe was diluted to 1:200 in hybridization buffer containing 50% deionized formamide, 10 mM Tris-HCl (pH 7.5), 2x SSC, 1x Denhardt’s solution, 2.5 mM dithiothreitol, 10% dextran sulfate, 400 µg/ml yeast tRNA, 1 mg/ml denatured and sheared salmon sperm DNA, and 0.5% SDS. Each section was overlaid with 100 µl diluted DIG-labeled RNA probe, covered with Parafilm, and incubated in a humid box containing 2x SSC and 50% deionized formamide at 50 C for 16–20 h. Then the slides were washed in 4x and 2x SSC at room temperature, 1x and 0.1x SSC at 42 C, and DIG wash buffer for 10 min, after incubation with blocking solution for 1 h (Roche Applied Science). The slides were incubated with anti-DIG-alkaline phosphatase (Fab fragments; Roche Applied Science) in a humidified box for 2 h. The slides were then rinsed in DIG wash buffer and developed in nitro blue tetrazolium chloride/5-bromo-4- chloro-3-indolyl phosphate, toluidine salt solution (NBT/BCIP ready-to-use tablets; Roche Applied Science) for 30 min. Thereafter, the sections were rinsed in DIG wash buffer and H2O, counterstained with nuclear fast red, and viewed via Zeiss Axioplan microscopy. A CoolSNAP CCD Camera (Photometrics Inc., Tucson, AZ) was used for image capture.

Western blotting
To analyze CRH/UCN-R-BP proteins in the macaque CL, Western blotting was performed as described previously (14) with some minor modifications. Briefly, CL proteins or positive controls (PC-12 plus NGF cell lysate for CRH, mouse heart extract for UCN3, U-87 MG cell lysate for CRHR2, and mouse liver extract for CRHBP from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; human stresscopin-related peptide for UCN2 from Phoenix Pharmaceuticals, Inc., Belmont, CA; and mouse embryo cell lysate for UCN, CRHR1 recombinant protein, from Abnova GmbH, Heidelberg, Germany) were incubated in the SDS-PAGE loading buffer at 95 C for 5 min. Samples were loaded onto a 4–20% Tris-HCl Ready Gel using a Mini-Protean II apparatus (Bio-Rad Laboratories, Hercules, CA). Proteins were transferred to a nitrocellulose membrane (Bio-Rad) overnight at 4 C. The membrane was blocked in 5% powered nonfat milk/Tris-buffered saline (Bio-Rad) for 1 h and then incubated overnight with primary rabbit antihuman antibodies [1:800 for CRH, 1:10 for UCN, and 1:4000 for loading control β-actin (Abcam Inc., Cambridge, MA); 1:400 for UCN2 (Phoenix Pharmaceuticals); and 1:100 for UCN3 and CRHBP (Santa Cruz Biotechnology); and 1:100 for CRHR1 and CRHR2 (Advanced Targeting Systems, San Diego, CA)] in blocking buffer while rocking at 4 C. An antirabbit IgG antibody conjugated with horseradish peroxidase (Zymed Laboratories, Inc., South San Francisco, CA) was used at a 1:4000 dilution. Antibody-protein complexes were visualized using an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ) and Kodak X-OMAT film (Eastman Kodak Co., Rochester, NY). Densitometry analysis was performed using a gel documentation system and Quantity One software (Bio-Rad).

In vivo protocol: intrafollicular injection and evaluation
Intrafollicular injection was performed on anesthetized monkeys 1 d before the midcycle E surge (d –1) as described previously (15, 16). Preliminary dose-response studies suggested that 5 µg/follicle astressin was the minimum dose that caused the serum E and P levels to drop during the luteal phase. There was no remarkable effect of a lower dose (1 µg/follicle) or greater effect of a higher dose (25 µg/follicle) for astressin on hormone levels (data not shown; n = 7). Therefore, the 5-µg/follicle dose was chosen for additional studies.

During a laparotomy to expose the ovary bearing the dominant follicle, a 28-gauge needle on an insulin syringe containing 50 µl solution containing either 10 µg CRHR antagonist astressin (Sigma-Aldrich, St. Louis, MO) or vehicle (0.1% BSA/PBS) was inserted through the stroma of the ovary before penetrating the follicular wall. Then, 50 µl follicular fluid was pulled into the syringe, diluting the injectable by half, before injecting 50 µl of this mixed solution into the follicle. The final delivery dose of astressin was 5 µg/follicle. Although vehicle treatment has little effect on ovulation and luteal development historically (15, 16), a sequential protocol was used typically with vehicle injected during the first experiment cycle (protocol 1); then after a cycle for rest and recovery, astressin was injected during the following experimental cycle (protocol 2). Ovaries (n = 5 per group) were evaluated by laparoscopy at 3 d after injection for evidence of ovulation (follicular stigmata formation). Images were recorded using the laparoscopic camera. Blood samples were collected on a daily basis throughout the expected luteal phase interval for 18 d or until menses and analyzed for serum E and P levels as described earlier.

In subsequent protocols, monkeys received an intrafollicular injection of either vehicle (n = 3) or the astressin (5 µg/follicle, n = 3) on d –1. On d 9 of the luteal phase, the ovary bearing the injected follicle was removed by laparoscopy and fixed in buffered formalin overnight before being embedded in paraffin (15, 16, 17). The paraffin-embedded tissues were serially sectioned at 5 µm and mounted on slides. A standard hematoxylin-eosin (H&E) staining was performed for general histological analyses (17). As a post facto addition, after the hormone and histological analyses that suggested subnormal luteal structure-function, nuclear DNA fragmentation in cells was detected using the DeadEnd colorimetric terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) system (Promega) following the manufacturer’s instructions with minor modifications as previously reported (18); these additional experiments were possible with available tissue sections. Slides were viewed via Zeiss Axioplan microscopy to evaluate luteal development of the injected follicle. A CoolSNAP CCD Camera (Photometrics) was used for image capture. TUNEL-positive cells were counted manually from three random areas of the CL for every slide.

Statistical analysis
Statistical evaluation of mean differences in protein levels (Western blotting densitometric analysis) among stages of the luteal phase was performed by one-way ANOVA, with a significance level set at 0.05 (SigmaStat, version 2.0; SPSS Inc., Chicago, IL). To identify significant differences between stages, the Student-Newman-Keuls post hoc test was used for pairwise multiple comparisons. P and E levels in serum were analyzed by ANOVA with repeated measures to identify differences between the controls and treatment group during the luteal phase (SigmaStat software). Differences were considered significant at P < 0.05, and values are presented as mean ± SEM. A Student’s t test was used to compare length of the luteal phase between control and astressin-treated animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CRH/UCN-R-BP mRNA localization in the monkey ovary
In the late follicular phase, there was minimal specific ISH staining (compared with negative controls; not shown) for CRH/UCN ligands in the preovulatory follicle, atretic antral follicles, or smaller preantral follicles (Fig. 1Go, A and B). However, specific staining for CRH, UCN, and UCN2 mRNAs was observed in the developing CL during the early luteal phase (Figs. 1CGo and 2AGo). CRH gene expression was also appreciable in the small preantral follicles in the ovarian cortex in the early luteal phase (Fig. 1CGo). In CL removed from the different stages of the luteal phase, CRH gene expression was apparent throughout the luteal phase (Fig. 2Go, A–C). In contrast, intense staining for both UCN mRNAs was most evident in the CL at early and mid-luteal phase (Fig. 2Go, A and B) but then appeared to decrease as the CL approached regression in the midlate luteal phase (Fig. 2CGo). The mRNA localized to the cytoplasm of granulosa-lutein cells of the CL (Fig. 2Go, A–C). No significant staining was evident in control sections processed with sense probes (bottom left of Fig. 2AGo; representative ECL section shown).


Figure 1
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  		FIG. 1. In situ staining for the mRNAs (blue) of CRH/UCN ligands in the rhesus macaque ovary containing the preovulatory follicle (A and B) and the ECL (C). Scale bars, 160 µm (A and C) and 20 µm (B). Cell nuclei are counterstained red. AF, Atretic follicle; F, antral follicle; G, granulosa cells; IT, interstitial tissue; OSE, ovarian surface epithelium; PAF, preantral follicle; POF, preovulatory follicle; T, theca cells.

 

Figure 2
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  		FIG. 2. In situ staining for the mRNAs (blue) of CRH/UCN ligands in the rhesus macaque CL during the early (ECL; A), mid (MCL; B), and midlate (MLCL; C) stage of the luteal phase. The nonspecific staining associated with the sense probe is illustrated in the inset of A. Cell nuclei are counterstained red. Bar, 20 µm.

 
During the late follicular phase, specific ISH localization of both CRHR (CRHR1 and CRHR2) mRNA was evident in theca cells of the preovulatory follicle and the surrounding stromal layer, with limited expression (notably for CRHR2) evident in the granulosa layer of the preovulatory follicle (Fig. 3Go, A and B). Appreciable staining for CRHR1 and CRHR2 was detected in the developing CL as well as interstitial cells in the ovarian stroma (Figs. 3CGo and 4AGo). CRH/UCN receptor staining was also observed in small preantral follicles during the early luteal phase but not the preovulatory follicular phase (Fig. 3Go, A and C). In contrast, ISH staining for CRHBP mRNA was not evident in small preantral and atretic follicles or interstitial tissue, whereas relatively limited staining was observed in both the granulosa and theca cells of the preovulatory follicle (Fig. 3Go, A and B) and in the developing CL during the early luteal phase (Figs. 3CGo and 4AGo). In CL isolated from the different stages of the luteal phase, the mRNA expression of CRHR was present at high levels from ECL to MCL (Fig. 4Go, A and B) before diminishing in the LCL (Fig. 4CGo). Although modest in situ staining for CRHBP mRNA was detectable early in the CL lifespan (Fig. 4AGo), minimal staining was observed in the MCL (Fig. 4BGo). The most intense CRHBP staining occurred in CL from the late luteal phase (Fig. 4CGo). Although receptor-specific staining appears to be concentrated in granulosa-lutein cells, CRHBP mRNA is present in both theca-lutein and granulosa-lutein cells (Fig. 4Go, A–C). Endothelial cells in the CL did not appear to stain intensely for CRH/UCN-R-BP mRNAs as in the luteal cells, which was consistent with our previous results on the lack of immunohistochemical staining for CRH/UCN-R-BP protein in the endothelial cells of the CL (8). However, additional studies are needed to confirm this issue and possible expression in immune cells. No receptor or binding protein staining was evident in control sections processed with sense probes (bottom left of Fig. 4AGo; representative ECL section shown).


Figure 3
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  		FIG. 3. In situ staining for the mRNAs (blue) of CRH/UCN receptors and binding protein in the rhesus macaque ovary containing the preovulatory follicle (A and B) and the ECL (C). Scale bars, 160 µm (A and C) and 20 µm (B). Cell nuclei are counterstained red. AF, Atretic follicle; F, antral follicle; G, granulosa cells; IT, interstitial tissue; OSE, ovarian surface epithelium; PAF, preantral follicle; POF, preovulatory follicle; T, theca cells.

 

Figure 4
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  		FIG. 4. In situ staining for the mRNAs (blue) of CRH/UCN receptors and binding protein in the rhesus macaque CL during the early (ECL; A), mid (MCL; B), and late (LCL; C) stage of the luteal phase. The nonspecific staining associated with the sense probe is illustrated in the inset of A. Cell nuclei are counterstained red. Bar, 20 µm.

 
CRH/UCN-R-BP protein levels in the macaque CL
As depicted in representative Western blotting (Fig. 5Go), the proteins for CRH, UCN, and UCN2 were expressed in the macaque CL throughout its lifespan in the menstrual cycle. UCN3 has previously been shown to be not expressed in the rhesus monkey ovary (8) and, therefore, was not analyzed for expression by Western blotting. Likewise, proteins for the CRHR1, CRHR2, as well as CRHBP, were also identified in macaque CL.


Figure 5
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  		FIG. 5. Mean (±SEM) protein levels for CRH/UCN-R-BP components relative to β-actin in the rhesus macaque CL throughout the luteal phase of the natural menstrual cycle. The total protein for Western blotting (represented at top of each panel) was generated from CL (n = 4 per stage) collected during the early (d 3–5 after LH surge), mid (d 6–8), midlate (d 10–12), late (d 14–16), and very late (d 18–19; menses) luteal phase. Values were standardized to respective β-actin protein control values. Columns with different letters are significantly (P < 0.05) different.

 
When normalized to β-actin (whose levels were invariant during the CL lifespan), CRH protein levels were high in the ECL and then decreased in the MCL (P < 0.05) (Fig. 5AGo). UCN protein levels were detectable throughout but decreased (P < 0.05) in the VLCL compared with ECL (Fig. 5BGo). Compared with the early-midlate stage, protein levels for UCN2 decreased significantly (P < 0.05) in the VLCL (Fig. 5CGo). CRHR1 protein levels were highest from the ECL to MLCL before gradually declining in the LCL to VLCL (P < 0.05) (Fig. 5DGo). CRHR2 protein levels decreased (P < 0.05) in the VLCL relative to MCL (Fig. 5EGo). In contrast, CRHBP levels were higher (P < 0.05) at the LCL than the other stages (Fig. 5FGo).

Effects of injection into the preovulatory follicle
All the control and astressin-treated monkeys (n = 5 per group) displayed a typical stigmata/rupture site on the ovary bearing the antecedent preovulatory follicle by 3 d after injection (Fig. 6Go, A and B). After the preovulatory surge, E levels declined by early luteal phase (d 2) in both groups. Thereafter, serum E concentrations increased gradually through the luteal phase in control monkeys, whereas the E levels of the astressin-treated monkeys remained at baseline and were diminished (P < 0.05) in midlate luteal phase (20–30 pg/ml) (Fig. 7AGo). Serum P concentrations increased in both control and astressin-treated monkeys during the early luteal lifespan (d 1–4). However, astressin administration significantly (P < 0.05) suppressed P levels during the mid (d 5) to midlate (d 13) stage of the luteal phase, compared with those in control monkeys (Fig. 7BGo). All the control monkeys began menses on d 17 of the luteal phase, whereas menstruation in astressin-treated monkeys began on d 13 ± 1.8 (P < 0.05) with two menstruating at d 8 and 9.


Figure 6
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  		FIG. 6. Ovarian indices of follicular rupture (protruding stigmata) by laparoscopic evaluation 3 d after intrafollicular injection with vehicle (0.1% BSA/PBS, A) and astressin (5 µg/follicle, B).

 

Figure 7
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  		FIG. 7. Serum E (A) and P (B) concentrations (mean ± SEM) during the luteal phase in rhesus macaques (n = 5 per group) receiving an intrafollicular injection (arrow) of 5 µg astressin compared with that of vehicle controls (0.1% BSA/PBS). m, Average length of luteal phase for astressin-treated animals; M, average length of luteal phase for the controls. *, Significant differences (P < 0.05) between groups in E or P concentrations on specific days.

 
On the day of ovariectomy (d 9), P levels were significantly lower in astressin-treated vs. control animals (2.3 ± 0.4 vs. 4.6 ± 0.3 ng/ml; n = 3; P < 0.05). As judged by H&E staining, luteal cells in the control CL displayed homogenous cytoplasm and round nuclei with very prominent nucleoli (Fig. 8AGo). The cytoplasm in the astressin-treated CL was vacuolated, and the nuclei were distorted with very few nucleoli (Fig. 8BGo). TUNEL staining was evident in large cells with brown nuclei (Fig. 8DGo, open arrows) and small cells with condensed fragmented nuclei (Fig. 8DGo, black arrows, TUNEL-positive cells) in the CL after astressin treatment. There were 3 ± 1 (mean ± SEM) TUNEL-positive cells per 100 cells. TUNEL-positive cells were also found in atretic follicles (Fig. 8EGo). In contrast, positive TUNEL staining was not found in any cell of the CL from animals receiving vehicle (BSA/PBS) (Fig. 8CGo) or in negative control slides (minus the TdT enzyme) (Fig. 8DGo) or in the surrounding stroma (Fig. 8EGo) where the nuclei were stained blue. There was an empty cavity lined by a single layer of fibroblastic cells in one CL after astressin treatment (Fig. 8FGo), whereas control CL were filled with luteal cells (not shown).


Figure 8
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  		FIG. 8. H&E staining (A and B) and TUNEL (C–F) of ovaries obtained from control (PBS/BSA, A and C) and astressin-treated (B and D–F) rhesus macaques on d 9 of the luteal phase. A–D and F, CL; E, atretic follicle. The negative control without the TdT enzyme is illustrated in the inset of F. TUNEL-positive cells were indicated by open arrows (large cells with brown nuclei) and black arrows (small cells with condensed nuclei, histological indices of cell degeneration). Scale bars, 40 µm (A and B), 20 µm (C–E), and 80 µm (F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report detailing the localization of CRH/UCN-R-BP mRNAs in the primate ovary during the preovulatory stage and luteal phase of the ovarian cycle. There was no obvious mRNA staining for ligands in the macaque ovary during the preovulatory phase. However, CRHR1 and CRHR2 mRNAs were detected in the theca and, to a lesser extent, the granulosa cells of the preovulatory follicle as well as the interstitial cells in the ovarian stroma. Thus, if the CRH/UCN-R system is active in the preovulatory follicle, it may require circulating ligands originating from outside the ovary. Notably, both ligand (CRH) and CRHR mRNAs were detected in small preantral follicles in the macaque ovary during the early luteal phase but not the preceding preovulatory stage. CRH and CRHR expression after the midcycle LH/FSH surge suggests that ligand-receptor action modulates early follicle growth. Previous studies focused on selected components of the CRH/UCN-R-BP system in developing antral follicles. For example, CRHR1 was localized to theca cells of human antral (7–8 mm in diameter) follicles (5) and the stroma and theca cells of the rat ovulatory follicles (19), which is consistent with our observations.

In contrast to the preovulatory follicle, both ligands (CRH, UCN, and UCN2) and receptor (CRHR1 and CRHR2) mRNAs were localized to the granulosa-lutein cells, but not theca-lutein cells, in the CL by the early luteal phase. This suggests that differentiating steroidogenic cells in the CL are the sites of CRH/UCN synthesis and CRHR-mediated action. With the exception of CRH, a high level of mRNA expression for the other ligands and receptors was observed in luteal cells in the early to mid stage of the luteal phase, suggesting that processes causing (e.g. the midcycle LH surge) or associated with (e.g. LH-induced local factors) follicle luteinization influence UCN-R expression. Subsequently, UCN and receptor staining declined in the CL during the later stages of the luteal phase. In general, the ISH localization of ligand/receptor mRNAs to luteal cells, particularly in the early to mid-luteal phase, is consistent with immunohistochemical localization of the corresponding proteins in the macaque CL (8).

In contrast, CRHBP mRNA was detected in both the theca and granulosa layer of the preovulatory follicle and the theca-lutein and granulosa-lutein cells of the CL, whereas CRHBP immunostaining was limited to only theca and theca-lutein cells (8). Because CRHBP is a secreted protein, it may be sequestered in the theca layer of the follicle or paraluteal region of the CL through unknown mechanisms. Unlike ligand-receptor mRNAs, CRHBP mRNA staining in the CL increased in the late luteal phase compared with other stages. Asakura et al. (5) 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 (7, 8), CRHBP is expressed in the macaque antral follicle and CL.

The Western blotting data were consistent with our previous RT- and real-time PCR results (8) indicating that all ligands, except UCN3 (data not shown), were expressed in the rhesus macaque CL. The expression of both receptors and the binding protein was also detected. The CRH/UCN-R-BP mRNAs (8) and proteins (current study) were dynamically expressed during the luteal lifespan in the menstrual cycle. CRH protein levels were high at the early stage of the luteal phase compared with the later stages, similar to our previous immunohistochemistry (IHC) results (8). CRH mRNA levels did not change, however, throughout the luteal lifespan according to real-time PCR (8) and current ISH analysis. Thus, the dynamic expression of CRH may be due to posttranscriptional or translational regulation. Both mRNA and protein levels of the UCNs and CRHRs were high at the early stage of the luteal phase before declining at later stages, which are supported by previous IHC (8) and current ISH results. The decrease in protein levels lagged to some extent behind the mRNA. For example, UCN and CRHR1 mRNA levels declined abruptly at midlate luteal phase (8), whereas protein levels did not decline significantly until the late to very late luteal phase. In contrast, the protein levels of CRHBP corresponded well with the mRNA data, which was supported by IHC and ISH analysis. Collectively, the data indicate that CRH/UCN and CRHR1/CRHR2 mRNA and protein expression peaked during either CL development or at the period of optimal P production (i.e. through the early to midlate stages). When CL regression was underway or complete (i.e. late to very late luteal phase), the expression of these proteins declined. In contrast, CRHBP mRNA and protein expression increased significantly in the CL during luteal regression. Previous studies demonstrated that significant CRH immunostaining was observed in developing CL and less prominent or totally absent in regressing CL of rodents and women (20, 21), which is consistent with our observations. However, Muramatsu et al. (6) reported higher levels of immunoreactive CRH and UCN in the regressing CL relative to the developing CL in women. This discrepancy may be due to the differences between species, methods for classifying stages of the luteal phase, and/or techniques employed.

The increased expression of CRH/UCN-CRHR1/CRHR2 in CL during early to mid-luteal phase suggests that this ligand-receptor system may promote CL structure and function. Evidence that the primary luteotropin LH increases UCN2 expression and suppresses CRHBP expression supports this hypothesis (8). Therefore, an in vivo study was designed to block local CRH/UCN-R action in the ovulatory follicle at midcycle to determine whether periovulatory events or subsequent CL development were altered. Astressin is a CRH-modified analog, a general antagonist with high affinity for both CRHR1 and CRHR2 (22). It has proven particularly potent at inhibiting CRH function in the hypothalamo-pituitary-adrenal axis in rhesus monkeys (23). Notably, injection of astressin into the preovulatory follicle 2–3 d before the expected time of ovulation had no effect on follicle rupture. This negative result contrasts with intrafollicular injection of antagonists to other ovarian local factors, e.g. prostaglandins (24) and angiopoietin (17), which blocked ovulation. Therefore, CRH/UCN ligands do not appear to regulate ovulation in monkeys.

The advantages of intrafollicular injection are, first, less drug is needed for the experiment, only several micrograms per treatment, and second, drug action can be considered primarily local, because its release into the bloodstream would quickly dilute its concentration and minimize systemic effects. Circulating CRH/UCN levels during the menstrual cycle of nonhuman primates have not been examined. Thus, the potential for a systemic negative or positive CRH/UCN feedback loop involving the developing CL cannot be ruled out. However, previous studies indicated that plasma concentrations of CRH remain lower than 2.1 pmol/liter throughout the estrous cycle in rodents (20) and also in women when CRH was highly expressed in the ovary (21). Exposure of the ovulatory and luteinizing follicle to the CRHR antagonist astressin did, however, reduce circulating E and P levels at the mid to midlate luteal phase of the menstrual cycle. Thus, CRH/UCN ligand-receptor action may promote luteal steroidogenic function.

Several reports indicate CRH can stimulate steroidogenesis in mammals. CRH increases estrogen biosynthesis in cultured human placental cells (25) and also stimulates, whereas CRH antagonist inhibits, P release in cultured rat granulosa cells (26). CRH treatment of mouse Leydig cell cultures stimulates steroidogenesis by increasing the synthesis of the steroidogenic acute regulatory protein that plays a critical role in facilitating the transfer of cholesterol across the mitochondrial membrane (27, 28). In human fetal adrenal cells, CRH induces cytochrome P450 cholesterol side chain cleavage (CYP11A) and 3β-hydroxysteroid dehydrogenase enzymes, both of which are necessary for P biosynthesis in the CL (29). There are also reports that CRH may suppress ovarian steroidogenesis. CRH exerts an inhibitory effect on estrogen and P production in cultured human granulosa-lutein cells (30, 31) and inhibited androgen production in isolated theca cells (32).

The presence of abnormal luteal cells and vacuolated structure in the midcycle CL after astressin treatment also suggests that CRH/UCN-R action promotes luteal development. Similar effects were observed in a previous study indicating preovulatory drug exposure (i.e. to an antiangiogenic agent) might disrupt subsequent luteal function (15). One of the hallmarks of luteinization is the further differentiation of the follicle wall into CL through tissue remodeling (33). A previous study indicated that CRH is a potent inducer of cell differentiation in fetal lung of baboons (34). In addition, the expression and activity of matrix metalloproteinases have been implicated in the remodeling of extracellular matrix that occurs in the CL throughout the cycle (12, 35). CRH and UCN were found to induce secretion of matrix metalloproteinases by cultured cells from human placenta and fetal membranes (36). Based on these findings and the results of the current study, CRH/UCN ligands may promote luteal formation and development perhaps via proteases.

Astressin treatment also shortened the luteal lifespan in some animals and appeared to promote luteal cell nuclear compaction and DNA degradation as observed by H&E and TUNEL analysis. This suggests that blocking CRH/UCN-R action promoted early luteolysis as early as mid-luteal phase. Increasing evidence supports the theory that apoptosis is an important mechanism controlling involution of the CL. Apoptotic indices were observed during structural involution of the CL in rodents and primates (18, 37, 38). In addition, CRH/UCN peptides reportedly protect diverse cell types from environmental insults, a so-called cytoprotective effect, which could maintain cell health and prevent apoptosis. CRH/UCN was proposed to provide protection to rat hippocampal neurons (39, 40) and cardiac myocytes from necrotic and apoptotic death (41, 42). CRH also acts as a cytoprotective agent in the Xenopus laevis tadpole tail with CRHBP blocking CRH action and hastening tail muscle cell death (43). Thus, CRH/UCN ligands may provide protection to luteal cells during luteal development and function at the early stage as a luteotropic factor. Subsequently, CRHBP neutralization of CRH/UCN bioactivity may promote luteal cell death through apoptosis during luteal regression. Although TUNEL staining is one accepted, but not definitive, marker of apoptosis, further quantitative analysis of cell death is required on additional tissues to rigorously evaluate the ability of CRH/UCN-R antagonists to promote apoptosis in the primate CL.

In summary, the current study provides detailed information regarding the specific ovarian cell types synthesizing CRH/UCN-R-BP components and the dynamic expression of the CRH/UCN-R-BP protein in the primate CL during the menstrual cycle. The pattern of the CRH/UCN-R-BP protein expression during the CL lifespan suggests that CRH/UCN regulate luteal processes primarily during the early to midluteal phase when there is greater expression of ligands/receptors and less expression of binding protein. By the late luteal phase, however, their luteal activities may be restricted because the expression of the ligands/receptors decreases and CRHBP increases. This is consistent with the hypothesis that ligand-receptor action promotes luteal development and/or structure-function, and its loss is associated with luteal regression. Evidence from direct injection of a CRHR antagonist into the preovulatory follicle supports the concept that the CRH/UCN-R system promotes luteal development and function but not the ovulatory process in primates. Whether the CRH/UCN-R-BP system acts directly or indirectly and which cellular processes are regulated by this system is unknown. Many issues remain to be resolved at the systemic, cellular, and molecular levels to understand the activities of the primate CL and its regulation by endocrine and local factors. The CHR/UCN-R-BP system may represent a novel regulatory system that functions in luteinization and luteolysis.


   Acknowledgments
 
We are grateful to members of the Division of Animal Resources as well as the members of ONPRC’s Endocrine Services Laboratory, Specialized Cooperative Centers Program in Reproduction and Infertility Research’s (U54) Molecular and Cell Biology Core, and Imaging and Morphology Core for their technical assistance.


   Footnotes
 
This study was supported by the National Institutes of Health Grants NIH NICHD HD20869 (R.L.S.) and NIH NICHD HD42000 (J.D.H.), through a cooperative agreement (U54 HD18185) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research, and the Primate Centers Grant NCRR RR00163.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 9, 2007

Abbreviations: CL, Corpus luteum; CRHR, CRH receptor; DIG, digoxigenin; E, estradiol; ECL, early CL; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ISH, in situ hybridization; LCL, late CL; MCL, mid-CL; MLCL, midlate CL; P, progesterone; SSC, standard saline citrate; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick end labeling; UCN-R-BP, urocortin-receptor-binding protein; VLCL, very late CL.

Received April 25, 2007.

Accepted for publication August 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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