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Endocrinology, doi:10.1210/en.2005-1372
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Endocrinology Vol. 147, No. 4 2018-2027
Copyright © 2006 by The Endocrine Society

Dynamic Changes in Mitogen-Activated Protein Kinase (MAPK) Activities in the Corpus Luteum of the Bonnet Monkey (Macaca radiata) during Development, Induced Luteolysis, and Simulated Early Pregnancy: A Role for p38 MAPK in the Regulation of Luteal Function

V. K. Yadav and R. Medhamurthy

Department of Molecular Reproduction, Development, and Genetics (V.K.Y., R.M.) and Primate Research Laboratory (R.M.), Indian Institute of Science, Bangalore 560012, India

Address all correspondence and requests for reprints to: Dr. R. Medhamurthy, Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore-560012, India. E-mail: rmm{at}mrdg.iisc.ernet.in.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Changes in MAPK activities were examined in the corpus luteum (CL) during luteolysis and pregnancy, employing GnRH antagonist (Cetrorelix)-induced luteolysis, stages of CL, and hCG treatment to mimic early pregnancy as model systems in the bonnet monkey. We hypothesized that MAPKs could serve to phosphorylate critical phosphoproteins to regulate luteal function. Analysis of several indices for structural (caspase-3 activity and DNA fragmentation) and functional (progesterone and steroidogenic acute regulatory protein expression) changes in the CL revealed that the decreased luteal function observed during Cetrorelix treatment and late luteal phase was associated with increased caspase-3 activity and DNA fragmentation. As expected, human chorionic gonadotropin treatment dramatically increased luteal function, but the indices for structural changes were only partially attenuated. All three MAPKs appeared to be constitutively active in the mid-luteal-phase CL, and activities of ERK-1/2 and p38-MAPK (p38), but not Jun N-terminal kinase (JNK)-1/2, decreased significantly (P < 0.05) within 12–24 h after Cetrorelix treatment. During the late luteal phase, in contrast to decreased ERK-1/2 and p38 activities, JNK-1/2 activities increased significantly (P < 0.05). Although human chorionic gonadotropin treatment increased ERK-1/2 and p38 activities, it decreased JNK-1/2 activities. The activation status of p38 was correlated with the phosphorylation status of an upstream activator, MAPK kinase-3/6 and the expression of MAPK activated protein kinase-3, a downstream target. Intraluteal administration of p38 kinase inhibitor (SB203580), but not MAPK kinase-1/2 inhibitor (PD98059), decreased the luteal function. Together, these data suggest an important role for p38 in the regulation of CL function in primates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORPUS LUTEUM (CL), a transient endocrine structure formed from the ruptured follicle after ovulation, is required for establishment and maintenance of pregnancy in mammals. In primates, the CL undergoes dynamic changes in structure and function during its short lifespan of 14–16 d, and circulating levels of LH are essential for its development, function, and survival (1, 2). Interestingly, however, spontaneous luteal regression that occurs at the end of the luteal phase of the nonfertile menstrual cycle does not appear to be the consequence of changes in circulating LH (3) or its receptor mRNA and protein levels (4). On the other hand, chorionic gonadotropin (CG), a functional analog of pituitary LH, emanating from the placental trophoblast extends the life span of the CL in fertile menstrual cycles, prompting investigators to suggest that changes downstream of the LH receptor underlie the cause for luteolysis. Accordingly, spontaneous luteolysis occurs as a consequence of progressively decreased responsiveness of the CL to the circulating LH (5, 6), but in the event of pregnancy, the process of luteolysis is overcome because of the higher circulating levels of CG (5, 7, 8).

LH and CG bind and activate the same G protein-coupled receptors on their target cells to primarily stimulate the adenylyl cyclase/cAMP/protein kinase A (PKA) pathway (1, 2, 9). Analysis of various components of the LH receptor downstream signaling cascade in luteal cells indicated that even though the activity of adenylyl cyclase appeared to be diminished during the late luteal phase (10), the activity of its principal downstream target, PKA, did not change significantly throughout the luteal phase (11). Although the presence of unvarying levels of PKA activity by nongenomic action might account for the maintenance of basal functions of luteal cells (12), the existence of other signaling pathway(s) that control luteal function during spontaneous luteolysis and pregnancy remains unclear. Several recent studies using single-gene expression analysis and high-throughput analyses, viz. microarray or differential display RT-PCR analysis, have observed that despite the constitutively activated PKA levels that account for nongenomic actions (11), changes in expression of a large number of genes occur during the processes of luteolysis and pregnancy (13, 14, 15, 16).

MAPKs represent a large family of kinases that respond to diverse physiological stimuli, viz. cell survival/proliferation, differentiation, and apoptosis. The mammalian MAPK superfamily comprises ERK, Jun N-terminal kinase (JNK)/stress-activated protein kinases, and p38 MAPK (p38). Activation of ERKs have been reported to be essential for survival/function and JNKs for cell death, whereas p38 has been reported to be important for survival/function as well as death in response to various growth factors or death stimuli in a cell-type-specific manner (17, 18). The hallmark of MAPK signaling is the regulation of transcriptional machinery by way of activation of transcription factors (19).

The mechanisms regulating the activation of MAPKs and the functional significance of their activation in the primate CL are unknown. Recent studies, however, have shown activation or inhibition of distinct subgroups of MAPKs in response to gonadotropins or prostaglandin F2{alpha} in the luteal cells of different species in vitro (20) and in vivo (21, 22), indicating that MAPKs may participate in the regulation of CL structure and/or function. The principal aim of the present study was to examine various components of MAPK pathways for evidence of regulation in response to changes in circulating levels of gonadotropins [LH/human CG (hCG)] employing GnRH antagonist-induced luteolysis, spontaneous luteolysis, and simulated early pregnancy model systems in the bonnet monkey.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
GnRH antagonist, Cetrorelix (CET) was a kind gift from Asta Medica (Frankfurt, Germany). hCG, Profasi, was from Ares Serono (Aubonne, Switzerland). Polyclonal antibodies specific to phospho-p38 (9211), phospho-stress-activated protein kinase/JNK (9251), phospho-p42/44 MAPK (9100), p38 (9212), ERK1 (sc-19), ERK2 (sc-154), JNK1 (sc-571), and JNK2 (sc-572) were purchased from Cell Signaling Technology (Beverly, MA) (9100, 9211, 9212, and 9251) and Santa Cruz Biotechnology Inc. (Santa Cruz), CA (sc-19, sc-154, sc-571, and sc-572). SB203580, PD98059, and caspase-3 substrate (264150) were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). ß-Actin antibody (A5441) was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Antibody against steroidogenic acute regulatory protein (StAR) was a kind gift from Professor D. M. Stocco (Texas Tech University Health Sciences Center, Lubbock, TX). Protein-A-agarose was purchased from Life Technologies, Inc. (Gaithersburg, MD). Phototope-HRP Western detection system with horseradish peroxidase-linked antirabbit IgG and p38 assay kit (9820) were purchased from Cell Signaling Technology. Terminal deoxynucleotidyl transferase was purchased from Amersham Pharmacia, Piscataway, NJ. Polyvinylidene difluoride (PVDF) and Genescreen membranes were purchased from NEN Life Science Products Life Sciences (Boston, MA). All other reagents were purchased from Sigma-Aldrich or Life Technologies or sourced locally.

Animal protocols and CL collections
Experimental protocols in the monkey were approved by the Institutional Animal Ethics Committee of the Indian Institute of Science. Adult female bonnet monkeys (Macaca radiata) weighing 3–5.2 kg with a history of regular menstrual cyclicity (27–29 d) were used for this study. The general care and housing of monkeys at the primate research laboratory, Indian Institute of Science, Bangalore, have been described elsewhere (23). During the experimentation period, the temperature in the animal rooms receiving continuous fresh 5-µm filtered air ranged from 22–28 C and 17–21 C, maximum and minimum, respectively. Studies were carried out during January to February and July to December months of each year, because female bonnet monkeys exhibit summer amenorrhea between the months of March and June (23). Blood samples through femoral venipuncture were collected daily from d 7 of the menstrual cycle until d 12 for determining the onset of estradiol (E2) and LH surges. Additional blood samples were collected either daily or on alternate days until the day of CL retrieval. In our colony, it was observed that the peak E2 and LH surges occurred simultaneously in 80% of the menstrual cycles monitored (10.33 d; n = 90). One day after the occurrence of the preovulatory LH peak was designated as d 1 of the luteal phase. The CL on the designated day of the luteal phase (see below) was retrieved from the ketamine hydrochloride-anesthetized [15 mg/kg body weight (BW)] monkeys subjected to laporotomy under aseptic conditions. Through careful dissection of the ovary, the CL was extirpated and transferred to a sterile petri dish containing filter paper, cut into quarters, placed in an individual sterile cryovial, and flash frozen in liquid nitrogen before storing at –70 C.

Changes in luteal structure and function were characterized based on the multiple criteria that are considered as markers of luteolysis and pregnancy in primates and other species, viz. serum progesterone (P4) (12) and luteal StAR mRNA and protein levels (14, 22, 24) for changes in luteal function (functional luteolysis) and caspase-3 activity (25, 26, 27) and low-molecular-weight (LMW) DNA fragmentation (27, 28) for changes in luteal structure (structural luteolysis).

Experiment I: temporal changes in CL structure/function during GnRH antagonist-induced luteolysis
It is well established that administration of GnRH antagonist into monkeys during the mid-luteal phase leads to inhibition of LH secretion and abrupt termination of the luteal phase (29, 30, 31). In the present study, CET was administered sc, at a dose of 75 µg/kg BW twice daily (0900 and 2100 h) on d 7 of the luteal phase of the menstrual cycle. In a pilot study, circulating serum P4 concentration decreased within 12 h and menstruation was initiated on d 3 or 4 after start of treatment at this dose. Twice-daily administration of CET was chosen because the half-life was reported to be 10 h (32). The GnRH antagonist was administered on d 7 of the luteal phase and CL (n = 3–4 per time point) collected from monkeys before and 12, 24, and 48 h after treatment to examine changes in MAPK activities after inhibition of pituitary LH secretion.

Experiment II: changes in CL structure/function during various stages of CL development and function
To determine the changes in CL structure/ function and to examine the MAPK activities during different stages of CL development/function, corpora lutea (n = 4 per stage) were collected from monkeys experiencing spontaneous menstrual cycles at early (d 5), mid (d 8–9), and late (d 14–15) luteal phase of the menstrual cycle.

Experiment III: changes in CL structure/function during simulated early pregnancy
To ascertain whether changes in MAPK activities play a role in the rescue of CL function after conception and establishment of pregnancy, a model system that simulates early pregnancy was employed. Studies carried out in rhesus (8) and bonnet (15) monkeys have demonstrated that exogenous administration of hCG in increasing doses beginning d 9 of luteal phase of the nonmated cycles mimics the circulating pattern of monkey CG, the steroid profile, and the expression profile of key steroidogenic enzymes in CL observed during early pregnancy. hCG was administered in increasing doses two times daily during 9–13 d of the luteal phase (15, 30, 45, 90, and 180 IU on d 9, 10, 11, 12, and 13 of the luteal phase, respectively), and CL (n = 3) was retrieved on d 14 of luteal phase. For comparison, CL collected on day 14 of the luteal phase, but without treatment, was used as untreated control.

Experiment IV: intraluteal administration of MAPK inhibitors on CL function
On d 9 of the luteal phase, i.e. during mid-luteal phase, vehicle (DMSO), p38 inhibitor (SB203580) or MAPK kinase (MEK)-1/2 inhibitor (PD985059) was administered directly into CL (n = 3 per vehicle or inhibitor) over a duration of 15 min. Earlier studies have reported the use of SB203580 in which intracerebroventricular administration of as little as 1 µg and systemic injections of 2.5 mg/kg BW in rats were used (33, 34). Because pharmacokinetic data in monkeys indicated higher doses with fewer side effects (35), administration of 500 µg of each inhibitor was chosen in the present study keeping in mind possible loss of inhibitor during infusion (see below). With the help of 100-µl Hamilton syringes, the inhibitors [dissolved in 50 µl dimethylsulfoxide (DMSO)] were infused in 5-µl aliquots directly into the central cavity region of CL as well as surrounding region of CL through the introduction of the syringe needle between the ovarian stroma and CL structure. During infusion, which lasted for 15 min, fluid and/or blood mixed with fluid was sometimes observed oozing out from CL and, consequently, loss of some infused inhibitor. For vehicle administration, 50 µl DMSO alone was administered intraluteally. Blood samples were collected before and 60 and 120 min after vehicle/inhibitor administration for estimation of serum P4 concentrations. To determine the specificity of p38 inhibition by SB203580 and MEK-1/2 inhibition by PD98059, corpora lutea collected 120 min after DMSO, SB203580, and PD98059 administration were subjected to Western blot analysis for pATF-2 (a downstream transcription factor phosphorylated by pp38; p38 inhibition) or phospho-ERK-1/2 (a downstream kinase phosphorylated by MEK-1/2; MEK-1/2 inhibition). To determine the effect of p38 and/or MEK-1/2 inhibition on steroidogenesis, CL tissue samples were subjected to RT-PCR analyses for StAR (as an index of steroidogenesis) and L-19 (housekeeping gene). To compare the P4 secretion pattern after lutectomy to that after inhibitor administration, in a separate experiment, three monkeys were subjected to lutectomy on d 8–9 of the luteal phase and blood samples collected before 2, 12, and 24 h after lutectomy.

Caspase-3 activity assays and DNA fragmentation analysis
Caspase-3 activity assays were carried out as described previously (27). Briefly, equal amounts (100 µg) of luteal lysate protein (prepared under nondenaturing conditions) from different treatments were added to 1 ml caspase-3 activity assay buffer [20 mM HEPES (pH 7.5), 10% glycerol, 2 mM dithiothreitol] followed by the addition of 20 µM Z-DEVD-AFC substrate. Reaction mixtures were incubated at 37 C for 1 h and liberated AFC was measured in a spectrofluorometer with an excitation wavelength of 400 nm and an emission wavelength of 420–520 nm. Specificity of the caspase-3 assay was determined by using a caspase-3-specific inhibitor (Ac-DEVD-CHO). Genomic DNA was extracted from individual CL, precipitated, dissolved in distilled water, and analyzed for quantification of LMW DNA fragments as described previously (22).

RNA isolation
Total RNA was extracted from luteal tissues using Trizol reagent according to the manufacturer’s recommendations. The quality and quantity of RNA sample were assessed spectrophotometrically. The OD 260:280 nm consistently gave a ratio of greater than 1.8.

Semiquantitative RT-PCR analysis
Semiquantitative RT-PCR was carried out essentially as described previously (15). Oligonucleotide primers were designed for StAR, MAPK activated protein kinase-3 (MAPKAPK-3) and L-19 based on the conserved regions in the mRNA of these genes. Primers (forward and reverse) and PCR conditions (annealing temperature and PCR cycle number) used were as follows: 5'-caaccaagagggctggaagaagg-3' and 5'-gtgggactccaggcgcttgc-3' for 564-bp StAR (60 C and 25 cycles); 5'-aggtagaccatcactggcag-3' and 5'-caacaggaggcggatcag-3' for 605-bp MAPKAPK-3 (52 C and 30 cycles); and 5'-gaaatcgccaatgccaactc-3' and 5'-tcttagacctgcgagcctca-3' for 406-bp L-19 (58 C and 23 cycles). Ethidium bromide-stained agarose gels displaying PCR products were scanned using UVI-Tech gel documentation system and quantitated using UVI-Band Map (1999) software (UVI-Tech Ltd., Cambridge, UK).

Preparation of tissue lysates and immunoblotting
CL tissue lysate was prepared following previously published procedures (22). Briefly, frozen CL tissue was homogenized in 200–300 µl RIPA buffer [10 mM NaPO4 (pH 7.0), 150 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 50 mM NaF, 200 mM Na3VO4, 0.1% ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, and 2 µg/ml leupeptin], transferred to Eppendorf tubes, and incubated on ice for 30 min with intermittent mixing before centrifugation at 15,000 x g for 10 min at 4 C. The clarified lysate was recovered, aliquoted, and stored at –70 C. The CL tissue lysate (100 or 200 µg protein) was resolved by 10% SDS-PAGE and electroblotted onto PVDF membrane using a semidry transfer unit (Bio-Rad Laboratories, Richmond, CA) as described previously (22). Western blot analysis was performed according to the published procedures (22). Autoradiographs were scanned using UVI-Tech gel documentation system and quantitated using UVI-Band Map (1999) software.

In vitro kinase assays
Assays were carried out in the linear range of the assay and with equal amounts of luteal lysate protein and antibodies as reported previously from this laboratory (ERK and JNK) (36) or according to the manufacturer’s protocol (p38). In brief, 100–200 µg CL tissue lysate protein was incubated with 20 µl of immobilized p38 antibody or phospho-JNK (1:200)/phospho-ERK (1:100) MAPK antibodies overnight followed by additional incubation with 20 µl protein-A-agarose for 3 h at 4 C. The resultant immune complexes were collected by centrifugation at 15,000 x g for 30 sec, and after washing, the immunoprecipitate was used directly in the assay. MAPK activities were assayed in the immune complexes using respective glutathione-S-transferase (GST) fusion proteins as substrates. For p38 activity assay, the immune complexes were washed in kinase buffer [25 mM Tris (pH 7.5), 5 mM ß-glycerophosphate, 10 mM MgCl2, 2 mM dithiothreitol, 0.1 mM Na3VO4], and the pellet was resuspended in 50 µl kinase buffer supplemented with 200 mM ATP and 2 µg ATF-2 fusion protein and incubated for 30 min at 30 C. The reaction was terminated by adding 25 µl of 3x SDS sample buffer. Samples were separated on a 10% acrylamide gel, transferred to PVDF membrane, and probed with phospho-ATF-2 antibody (1:1000). For phospho-JNK and ERK kinase activity assays, the immune complexes were washed in the kinase buffer and the pellet resuspended in 25 µl kinase buffer supplemented with 20 µM ATP, 2.5 µCi [{gamma}32P]ATP and 2 µg GST-c-Jun (phospho-JNK assay) or Elk-1 (phospho-ERK assay) fusion proteins and incubated for 30 min at 30 C. The reaction was terminated by adding 12.5 µl of 3x SDS sample buffer, and samples were separated on a 12% acrylamide gel, followed by gel drying and autoradiography.

Hormone assays
E2 and P4 in serum were determined by specific RIAs as reported previously (37). The E2 (GDN no. 244) and P4 (GDN no. 337) antisera were kindly provided by Professor G. D. Niswender (University of Colorado, Fort Collins, CO).

Statistical analyses
Data where applicable are expressed as mean ± SEM. The arbitrary densitometric units were represented as the percentage relative to control, which was set at 100%. The data were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test (PRISM Graph Pad version 2; Graph Pad Software Inc., San Diego, CA). A P value of <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of CL function during CET-induced luteolysis, different stages, and simulated early pregnancy
Figure 1Go illustrates serum P4 concentrations (A) and steady-state levels of StAR mRNA and protein expression (B and C) in the CL during CET treatment, different stages of luteal phase, and simulated early pregnancy. Serum P4 concentrations decreased significantly (P < 0.05) within 12 h after CET injection and remained low throughout the treatment period (Fig. 1AGo). This decrease in serum P4 was associated with a significant decrease in the StAR mRNA and protein levels in the CL (Fig. 1Go, B and C). Serum P4 concentrations were maximal during the mid-luteal phase (Fig. 1AGo) associated with high (P < 0.05) StAR mRNA and protein expression (Fig. 1Go, B and C), but the levels decreased significantly (P < 0.05) at the late luteal phase (Fig. 1Go, A–C). However, hCG treatment initiated at the mid-luteal phase significantly (P < 0.05) increased the luteal function as reflected in the serum P4 levels (Fig. 1AGo) and StAR mRNA and protein expression (Fig. 1Go, B and C) in the CL at d 14 of the luteal phase.


Figure 1
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  		FIG. 1. Changes in serum P4 and StAR mRNA and protein levels. A, Mean (± SEM) serum concentrations of P4 before and after CET injection, different stages of CL (early, mid, and late luteal phase) and after exogenous hCG treatment on d 9–13 of the luteal phase to simulate early pregnancy (CL collected on d 14). Bars with different letters are significantly different (P < 0.05). B, RNA isolated from the luteal tissue collected before and after CET treatment, during different stages (early, mid, and late luteal phase) and after exogenous hCG treatment were reverse transcribed and analyzed for steady-state changes in StAR and L-19 mRNA levels by semiquantitative RT-PCR analysis (top panels). Tissue lysates (100 µg) prepared from corpora lutea collected from monkeys that received different treatments (see above) were resolved on SDS-PAGE, transferred to PVDF membrane, and probed with StAR antibody; blots were stripped and reprobed with ß-actin antibody (bottom panels). Representative ethidium bromide-stained agarose gels of semiquantitative RT-PCR analysis of StAR and L-19 expression and immunoblots of StAR protein and ß-actin levels in CL are shown. C, The levels of StAR mRNA (open bars) and protein levels (solid bars) as determined in Fig. 1BGo from time 0 h (CET treatment), mid-luteal phase (stages of CL), and late luteal phase without hCG treatments relative to L-19 mRNA levels (for StAR mRNA) and ß-actin levels (for StAR protein levels). Bars with different letters are significantly different (P < 0.05). E, Early luteal phase; M, mid-luteal phase; L, late luteal phase.

 
Caspase-dependent cell death during different functional status of the monkey CL
Figure 2Go illustrates changes in the LMW DNA fragmentation (top panels) and caspase-3 activity (bottom panels) during different functional status of CL as characterized based on the serum P4 and StAR mRNA and protein levels in the CL. Although some evidence for DNA fragmentation was observed in the untreated CL, the LMW DNA fragments increased significantly (P < 0.05) within 12 h of CET treatment and remained high throughout the experimental period. The increased DNA fragmentation observed during CET treatment was associated with increased caspase-3 activity. Compared with the early luteal phase, LMW DNA fragments and caspase-3 activity were higher (P > 0.05) at the mid-luteal phase, but the levels increased (P < 0.05) further to reach more than 2-fold in the late luteal phase CL. However, exogenous hCG treatment during 9–13 d of the luteal phase attenuated the incidence of LMW DNA fragments in the CL collected on d 14 of the luteal phase, whereas the caspase-3 activity was only marginally decreased after hCG treatment.


Figure 2
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  		FIG. 2. Caspase-3 (Cas-3) activity assay and DNA fragmentation analysis. Caspase-3 activity (top panel) and DNA fragmentation levels (bottom panel) at time 0 h (CET treatment), early luteal phase (stages of CL), and late luteal phase without hCG treatment were set as 1-fold (control), and all other values within each experiment were calculated as fold change from control. Caspase-3 activity was assayed using Z-DEVD-AFC as a substrate. The quantitative measurement of LMW DNA labeling was determined from CL in each experiment. The values are expressed as mean ± SEM; n = 3 CL per time point. Bars with different letters are significantly different (P < 0.05). E, Early luteal phase; M, mid-luteal phase; L, late luteal phase.

 
Changes in the MAPK activation status during different functional status of the monkey CL
Analysis of luteal tissue lysates for changes in ERK-1/2 activation during CET-induced luteolysis revealed that the levels decreased significantly (P < 0.05) within 24 h after CET treatment and remained low thereafter (Fig. 3Go, A–C). Similar to the ERK-1/2 activation, p38 activation levels were also high in CL collected from the mid-luteal phase but decreased (P < 0.05) within 12 h of CET treatment to reach less than 25% of control levels at 48 h (Fig. 3Go, A–C). Although JNK-1/2 activation level was high in CL from the mid-luteal phase, CET treatment had no significant (P < 0.05) effect on the activation levels (Fig. 3Go, A–C).


Figure 3
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  		FIG. 3. Changes in activation levels of MAPKs during CET treatment. A, Tissue lysates prepared from CL collected before and at different times after CET injection were resolved on SDS-PAGE, transferred to PVDF membrane, and probed with phospho-MAPK antibodies; the blot was stripped and probed with total MAPK antibodies. The blots shown are from one of three independent experiments (CL from one animal from each time point was used per experiment). B, In vitro MAPK assays. Tissue lysates were subjected to immunoprecipitation with phospho-MAPK antibodies followed by in vitro phosphorylation of GST-fusion proteins as described in Materials and Methods. The relative level before CET treatment (time 0 h) was set as 1, and all other values for each kinase analysis, expressed in relation to the before-treatment value of 1, are shown below the blots. C, Immunoblots for phospho-MAPKs and total MAPKs were analyzed by densitometry, and the relative amounts of phospho-/total MAPKs compared with before treatment (control) are depicted graphically. Bars with different letters are significantly different (P < 0.05).

 
Changes in MAPK activation levels during different stages of CL are presented in Fig. 4Go. ERK-1/2 activation levels were similar in early and late CL but were higher (P < 0.05) during the mid-luteal phase (Fig. 4Go, A–C). Similar to the ERK-1/2 activation, p38 activation levels were significantly high (P < 0.05) in the mid CL, compared with the levels in early and late CL (Fig. 4Go, A–C). JNK-1/2 activation levels were significantly high (P < 0.05) in the CL collected during the mid and late luteal phases compared with the early CL (Fig. 4Go, A–C).


Figure 4
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  		FIG. 4. Changes in activation levels of MAPKs during different stages of luteal phase. A, Tissue lysates (100 µg) prepared from corpora lutea collected at different stages (early, mid, and late luteal phase) were resolved on SDS-PAGE, transferred to PVDF membrane, and probed with phospho-MAPK antibodies; blots were stripped and probed with total MAPK antibodies. The blots shown are from one of three independent experiments (CL from one animal from each time point was used per experiment). B, In vitro MAPK assays. Tissue lysates were subjected to immunoprecipitation with phospho-MAPK antibodies followed by in vitro phosphorylation of GST-fusion proteins as described in Materials and Methods. The relative level at mid-luteal phase was set as 1, and all other values for each kinase analysis, expressed in relation to the before-treatment value of 1, are shown below the blots. C, Immunoblots for phospho-MAPKs and total MAPKs were analyzed by densitometry, and the relative amounts of phospho-/total MAPK compared with controls (see Fig. 1Go for details) are depicted graphically. Bars with different letters are significantly different (P < 0.05). E, Early luteal phase; M, mid-luteal phase; L, late luteal phase.

 
Analysis of MAPK activation levels in CL of monkeys that received hCG treatment during d 9–13 of the luteal phase are presented in Fig. 5Go. ERK-1/2 and p38 activation levels were significantly higher (P < 0.05) in the CL from the hCG-treated monkeys compared with the age-matched CL from untreated monkeys (Fig. 5Go, A–C). However, JNK activation levels were lower (P < 0.05) in CL from monkeys that received hCG treatment compared with the levels in the age-matched CL from untreated monkeys (Fig. 5Go, A–C).


Figure 5
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  		FIG. 5. Changes in activation levels of MAPKs after exogenous hCG treatment. A, Tissue lysates (100 µg) prepared from corpora lutea collected at d 14 of luteal phase without (–) or with (+) hCG treatment (from d 9–13 of luteal phase) were resolved on SDS-PAGE, transferred to PVDF membrane, and probed with phospho-MAPK antibodies; blots were stripped and probed with total MAPK antibodies. The blots shown are from one of three independent experiments (CL from one animal from each time point was used per experiment). B, In vitro MAPK assays. Tissue lysates were subjected to immunoprecipitation with phospho-MAPK antibodies followed by in vitro phosphorylation of GST-fusion proteins as described in Materials and Methods. The relative level without hCG treatment (–) was set as 1, and all other values for each kinase analysis, expressed in relation to the before-treatment value of 1, are shown below the blots. C, Immunoblots for phospho-MAPKs and total MAPKs were analyzed by densitometry, and the relative amounts of phospho-/total MAPK compared with controls (see Fig. 1Go for details) are depicted graphically. Bars with different letters are significantly different (P < 0.05).

 
Changes in phospho MAPK kinase 3/6 (MKK-3/6) and MAPKAPK-3 mRNA levels correlate with changes in p38 activation
We investigated whether the kinetics of p38 activation is correlated with the activation levels of its upstream kinase, MKK-3/6, using an antibody that recognizes phosphorylated MKK-3/6 (Fig. 6AGo), and changes in the mRNA expression of one of the downstream targets of p38, MAPKAPK-3, by semiquantitative RT-PCR analysis (Fig. 6BGo). The phosphorylation levels of MKK-3/6 were high in the mid-luteal phase CL but decreased significantly (P < 0.05) within 12 h after CET treatment and remained low throughout the treatment period (Fig. 6AGo). The phospho-MKK-3/6 levels were lower (P < 0.05) during early and late luteal phases compared with the mid-luteal phase (Fig. 6AGo). Treatment with hCG resulted in increased phosphorylation of MKK-3/6 (Fig. 6AGo). During CET treatment, the expression of MAPKAPK-3 decreased significantly (P < 0.05) within 24 h to reach less than 20% at 48 h (Fig. 6BGo). The MAPKAPK-3 expression was significantly (P < 0.05) higher in the CL from the mid-luteal phase compared with the early and late luteal phase. Treatment with hCG resulted in increased expression of MAPKAPK-3 (Fig. 6BGo).


Figure 6
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  		FIG. 6. Analysis of upstream and downstream components of p38 signaling module in the corpus luteum. A, Tissue lysates (100 µg) prepared from corpora lutea collected from monkeys in different experiments were resolved on SDS-PAGE, transferred to PVDF membrane, and probed with phospho-MKK-3/6 antibody. The blots shown are from one of three independent experiments. Immunoblots were analyzed by densitometry, and the relative levels before CET treatment (time 0 h), mid-luteal phase (stages of CL), and late luteal phase (hCG treatment) were set as 1; values at other time points in each experiment are expressed relative to 1. Bars with different letters are significantly different (P < 0.05). B, Representative ethidium bromide-stained agarose gels of semiquantitative RT-PCR analyses of MAPKAPK-3 and L-19 in CL obtained from monkeys from different experiments. The stained agarose gels were analyzed by densitometry, and the relative levels at time 0 h (CET treatment), at mid-luteal phase (stages of CL), and at late luteal phase (without hCG treatment) were set as 1; values at other time points in each experiment are expressed relative to 1. Bars with different letters are significantly different (P < 0.05). E, Early luteal phase; M, mid-luteal phase; L, late luteal phase.

 
Effect of intraluteal infusion of specific MAPK inhibitors
Because ERK-1/2 and p38 kinase activities correlated positively with the changes in luteal function, we further investigated the role of these kinases in the regulation of luteal function with the use of specific inhibitors in vivo. Changes in the endogenous levels of phospho-ATF-2, in StAR mRNA expression, and in the serum P4 in monkeys after vehicle or SB203580 (p38 inhibitor) treatment are presented in Fig. 7Go, A–C. We first analyzed changes in ATF-2 phosphorylation (a downstream target of pp38) to confirm in vivo inhibition of p38 activity, and the results indicated that SB203580 treatment decreased pATF-2 (Fig. 7AGo). This decrease in p38 activity was associated with a decrease in StAR mRNA expression in the CL (Fig. 7BGo). Serum P4 concentrations decreased approximately 40% in monkeys receiving SB203580 treatment (Fig. 7CGo). However, as can be seen in Fig. 7Go, D–F, although a MEK-1/2 inhibitor (PD98059) specifically inhibited MEK-1/2 activity leading to decreased pERK-1/2 levels (Fig. 7DGo), no significant changes in StAR mRNA expression (Fig. 7EGo) or circulating P4 were seen (Fig. 7FGo). In another experiment, lutectomy on d 8–9 of the luteal phase resulted in approximately 70% circulating P4 levels within 2 h and continued decline to reach the lowest levels at 24 h (serum P4 levels before and 2 and 24 h after lutectomy were 2.53 ± 0.19, 0.7 ± 0.12, and 0.18 ± 0.09 ng/ml, respectively). Compared with the approximately 70% decrease in P4 levels within 2 h after lutectomy, there was an approximately 40% decrease in P4 levels observed 2 h after intraluteal administration of SB203580.


Figure 7
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  		FIG. 7. Effects of MAPK inhibitors SB203580 (SB) and PD98059 (PD) on luteal function in vivo. A–C, Effect of inhibition of p38 activity by SB. A, Western blot analysis of changes in phospho-ATF-2 levels in luteal tissue lysates (100 µg) prepared from CL collected before and at 120 min after intraluteal administration of vehicle (DMSO) or SB (500 µg). Blot was stripped and probed with ß-actin antibody. B, Semiquantitative RT-PCR analysis of StAR mRNA from CL collected before and at 120 min after intraluteal administration of vehicle (DMSO) or SB. L-19 is shown as an internal control. C, Mean (± SEM) serum P4 levels before and 60 and 120 min after intraluteal administration of DMSO or SB. D–F, Effect of inhibition of MEK-1/2 activity by PD. D, Western Blot analysis of changes in phospho-ERK-1/2 levels in luteal tissue lysates (100 µg) prepared from CL collected before and at 120 min after intraluteal administration of vehicle (DMSO) or PD (500 µg). Blot was stripped and probed with total ERK-1/2 antibody. E, Semiquantitative RT-PCR analysis of StAR mRNA from CL collected before and at 120 min after intraluteal administration of vehicle (DMSO) or PD. L-19 is shown as an internal control. F, Mean (± SEM) serum P4 levels before and 60 and 120 min after intraluteal administration of DMSO or PD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite the fact that luteal phase defects contribute to a high incidence of early pregnancy losses reported in humans (38), the molecular mechanisms that regulate dynamic changes in the luteal structure and function are far from clear. Previous studies have demonstrated an indispensable role for LH receptor signaling in the maintenance of luteal function; however, the downstream signaling components that underlie changes in luteal function especially during luteolysis and early pregnancy in primates remain unclear. In the present study, we have investigated changes in the activation status of MAPKs during different stages of the luteal phase, induced luteolysis, and hCG-simulated early pregnancy. The results showed that decreased luteal function observed after induced luteolysis was associated with decreased activation levels of p38 and ERK, but the activation status of JNK remained unaltered. The findings prompted us to investigate whether similar changes in MAPK activation levels would also occur during decreased luteal function that occurs during the late luteal phase. Indeed, p38 and ERK activation levels were lower from the CL collected at d 14 of the luteal phase of the nonfertile cycle, but treatment of exogenous hCG to mimic early pregnancy prevented decrease in the activation level of MAPKs. In contrast, JNK activation levels were higher at the late luteal phase, but the levels decreased after hCG treatment.

The results of the present study suggest that higher p38 activation levels correlated with higher luteal function in the bonnet monkey and that the changes in p38 activation status were associated with changes in the activation levels of MKK-3/6, an upstream activator of p38, and the expression levels of one of the downstream targets, MAPKAPK-3. Although p38 signaling is generally considered to be activated in response to environmental stresses (17, 18), more recent studies have shown that activation of p38 can also lead to other biological outcomes such as proliferation, cell survival, and differentiation depending on the context and cell type (17, 39, 40). Changes in p38 activation status during the luteal maturation have been studied in the rat. It was reported that a p38 downstream protein kinase target, MAPKAPK-3, was induced at both mRNA and protein levels during luteal maturation, and inhibitor studies in a cellular model of luteinization suggested requirement of an intact p38 path for phosphorylation of cAMP response element binding protein (21). In the monkey CL, whether the activated p38 path will also activate the downstream targets such as cAMP response element binding protein remains to be determined.

To further understand the relationship between activated p38 and ERK levels and luteal function, we have examined the effect of intraluteal administration of specific p38 and ERK inhibitors on luteal function during the mid-luteal phase. Direct effects of intraluteal infusion of MAPK inhibitors on luteal function were assessed during the acute treatment period for the following reasons. First, in the pilot study, intraluteal administration of SB203580 revealed changes in the phosphorylation status of ATF-2 within 2 h. Second, in our experience, there appears to be two distinct phases of P4 decrease after CET treatment with an initial decrease beginning at 2 h followed by a more profound decrease approximately 24 h later. Moreover, in a recent experiment, it was observed that pp38 level increased within 60 min after exogenous administration of a recombinant human LH preparation coincident with increased circulating P4 levels in monkeys treated previously with CET for 3 h (41). Intraluteal administration of p38 inhibitor resulted in decreased StAR mRNA levels in the CL, and this decrease was associated with a lower serum P4 level. The p38 inhibitor experiment together with our observation that a higher p38 activation level was associated with higher luteal function during different functional status of the CL suggest a positive role for the p38 pathway in the regulation of P4 production by the monkey CL. The observation of a positive role for p38 in the regulation of steroidogenesis via regulation of StAR expression has previously been reported by others. Svechnikov et al. (42) observed that IL-1{alpha}-stimulated StAR gene expression was dependent on the p38 pathway in immature Leydig cells, and the stimulatory effect of IL-1{alpha} could be abolished by pretreatment with SB203580. In the present study, we cannot rule out the possibility that SB203580 also targeted other kinases in addition to p38. Although SB203580 is considered to be selective in its action (40, 43), it has recently been reported that SB203580 inhibits JNK2-related isoforms in the cardiac tissue, albeit at a higher IC50 than required for inhibition of p38 (44). In the present study, it was unlikely that inhibition of JNK accounted for decreased steroidogenesis observed in response to SB203580 treatment, because alterations of JNK activation status did not accompany the decreased steroidogenesis that was seen in response to CET treatment, even though lower JNK activation was observed after hCG-stimulated steroidogenesis.

Other studies have found that LH/hCG-induced changes in the ERK MAPKs play an important role in determining the survival status or function of a variety of ovarian cell types including luteal cells. Cameron et al. (45) and Das et al. (46) independently reported that ERK1/2 was activated by FSH and LH in a cAMP/PKA-dependent manner in porcine and rat granulosa cells. In studies employing human luteinized granulosa cells, Dewi et al. (20) observed that LH/hCG increased activation of ERK and its upstream activator, MEK-1/2. Although activation of ERK was observed within minutes, a sustained activation lasting more than 24 h after exposure to the gonadotropin was also observed. It has been reported that MAPK pathways can mediate distinct cellular responses depending on whether the activation of the pathway is transient (occurring within minutes) or sustained (lasting for hours) (47). In human granulosa-lutein cells, LH/hCG-induced activation of ERK could be inhibited by the pharmacological inhibitors of MEK, but LH/hCG- and 22R-hydroxy cholesterol-stimulated P4 production remained unaffected (20). However, cholera toxin-, forskolin-, and (Bu)2cAMP-induced ERK activation as well as increase in P4 production in granulosa-lutein cells were blocked by MEK inhibition, indicating perhaps that ERK plays a role in regulating P4 synthesis independent of LH stimulation (20). Salvador et al. (48) also did not observe changes in steroidogenesis after acute inhibition of PKA-dependent ERK activity by PD98059. In the present study, intraluteal administration of PD98059 also decreased activation status of ERK as reported for granulosa-lutein cells, but this was not associated with a decrease in P4 secretion. It should be pointed out that in the present study, the effects of MEK inhibition were not examined during the gonadotropin-stimulated condition to determine whether ERK activation has a role in steroid biosynthesis. In contrast to the above observations, inhibition of MEK appears to restore LH-stimulated P4 production inhibition by the platelet-derived growth factor in porcine thecal cells (49), and MEK inhibition has been reported to enhance steroid biosynthesis in FSH-stimulated porcine granulosa cells (50), indicating that depending on the cell type and stimulus employed the activation of ERK may have a different role.

In summary, we have examined the activation status of MAPKs during different functional status of CL in the monkey. Although the activation status of p38 correlates positively with the functional status of CL, whether there exists a relationship between ERK and JNK activation levels and the functional status of monkey CL requires additional investigation involving the use of appropriate in vitro and in vivo model systems.


   Acknowledgments
 
We are grateful to Prof. D. M. Stocco (Texas Tech University Health Sciences Center, Lubbock, TX) for kindly providing the StAR antibody, Dr. S. G. Ramachandra of Primate Research Laboratory for assistance with surgery, and Mrs. G. Lakshmi and Mr. P. Jayaram for help in the preparation of this manuscript.


   Footnotes
 
V.K.Y. was supported by a fellowship from the Council of Scientific and Industrial Research, New Delhi, India. This work was supported in part by grants from the Council of Scientific and Industrial Research, Indian Council of Medical Research, and University Grants Commission, New Delhi, India.

Portions of this work were presented in abstract (P2-415) form at the 84th Annual Meeting of the Endocrine Society, San Francisco, CA, 2002.

V.K.Y. and R.M. have nothing to declare.

First Published Online January 12, 2006

Abbreviations: BW, Body weight; CET, Cetrorelix; CG, chorionic gonadotropin; CL, corpus luteum; DMSO, dimethylsulfoxide; E2, estradiol; GST, glutathione-S-transferase; hCG, human CG; LMW, low-molecular-weight; MAPKAPK, MAPK activated protein kinase; MEK, MAPK kinase; MKK-3/6, MAPK kinase-3/6; P4, progesterone; p38, p38-MAPK; PKA, protein kinase A; PVDF, polyvinylidene difluoride; StAR, steroidogenic acute regulatory protein.

Received October 28, 2005.

Accepted for publication January 5, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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