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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-2214
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2327-2335
Copyright © 2005 by The Endocrine Society

Dynamic Expression of Caspase-2, -3, -8, and -9 Proteins and Enzyme Activity, But Not Messenger Ribonucleic Acid, in the Monkey Corpus Luteum during the Menstrual Cycle

Marina C. Peluffo, Kelly A. Young and Richard L. Stouffer

Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Studies were designed to determine whether: 1) changes in caspase expression or activity occur in the macaque corpus luteum (CL) during its lifespan in the menstrual cycle, and 2) LH acting directly or via ovarian steroids regulates luteal caspases. Caspase-2, -3, -8, and -9 mRNAs were detectable by semiquantitative RT- or real time-PCR in CL, but levels did not differ between the early, mid, mid-late, late, and very-late luteal phases. Immunostaining for caspase-2 and -3 proteins was observed in luteal cells and appeared to peak by mid to mid-late stage. Enzyme activity for caspase-2, -3, -8, and -9 increased (P < 0.05) by mid-late stage, and then declined by the very-late stage. Treatment with GnRH antagonist + LH at the mid-late stage increased caspase-2, -8, and -9, but not -3, activity, compared with controls. Coadministration of a steroid synthesis inhibitor (trilostane) with GnRH antagonist + LH reduced (P < 0.05) caspase-2, -8, and -9 activity. Progestin (R5020) replacement during trilostane treatment did not restore caspase activity. Thus, initiator and effector caspases are present during CL development and regression in the menstrual cycle. The increased caspase activity at mid-late stage suggests that apoptosis is involved in early luteolysis in primates. Gonadotropin, perhaps via local steroids, modulates initiator caspases in the primate CL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CORPUS LUTEUM (CL) forms from the somatic cells of the follicle after ovulation and secretes progesterone (P), as well as other hormones, that permit the initiation and maintenance of intrauterine pregnancy (1). If pregnancy does not occur, the CL ceases to function and undergoes structural regression. In many primates, from Old World monkeys to great apes and women, CL development, function, and regression occurs over a 2-wk interval during the latter half of the menstrual cycle. The midcycle surge of LH secreted by the pituitary gland, pulsatile LH secretion during the luteal phase, and chorionic gonadotropin (CG) secretion after implantation play critical luteotropic roles in regulating the development, function, and lifespan of the primate CL (2). However, recent evidence suggests that LH/CG actions are mediated at least in part, via locally produced factors, including the steroid hormone P (2). The formation and regression of the CL involves extensive tissue remodeling, and investigators recently identified several proteases (3), including members of the matrix metalloproteinase family (4, 5) whose expression in the ovulatory, luteinizing follicle or CL is regulated by P.

The cellular and molecular processes responsible for the tissue remodeling associated with CL formation and regression is an area of active investigation (6, 7). Considerable evidence suggests that programmed cell death, or apoptosis, is associated with the physiological remodeling occurring in the ovary (8). Apoptosis, a process initiated when cells are damaged or no longer needed, follows a stereotypical pattern of biochemical and morphological changes that requires the up- or down-regulation of certain genes or gene products. Based on research in several model systems, members of the caspase (cysteine aspartic acid-specific protease) family are believed to contribute to the initial (e.g. initiator caspases 2, 8, 9, 10) and final (e.g. effector caspases 3, 6, and 7) apoptotic events (9, 10, 11). In the CL, apoptosis appears associated with luteal regression in many species, including rodents and domestic animals (12, 13, 14, 15, 16, 17). However, there is some controversy about the involvement of apoptosis during luteolysis in primates (for review, see Ref. 18). In addition, there are few studies to date on caspase expression or activity in the ovary or CL (18, 19, 20). Recent evidence suggests that caspase 3 is expressed in human luteinized granulosa cells (21) and CL (22, 23). Studies in caspase 3-deficient mice (24) suggest that this enzyme is pivotal for the timely regression of the CL.

We hypothesized that if apoptosis is involved in the tissue remodeling associated with the development or regression of the primate CL, then caspase mRNA expression, protein levels, and/or enzyme activity would be associated with the early or later stages of the luteal lifespan in the menstrual cycle. In addition, we hypothesized that alterations in luteotropic (LH) support may directly, or indirectly via P or other steroids, modulate caspase expression or activity. Therefore, studies were designed to examine and compare caspase levels and activity in the CL of rhesus monkeys: 1) at specific stages during the luteal lifespan in the natural menstrual cycle, and 2) after gonadotropin and/or steroid hormone ablation and replacement at mid-to-late luteal phase. In these initial studies, we focused on two initiator caspases (-2 and -8) involved in death receptor-initiated apoptosis, one caspase (-9) involved in mitochondrial-initiated apoptosis, and the general downstream effector, caspase-3 (8).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and protocols

The general care and housing of monkeys (Macaca mulatta) at the Oregon National Primate Research Center (ONPRC) was described previously (25). The ONPRC Animal Care and Use Committee approved all animal protocols and experiments, and studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The menstrual cycle of adult female rhesus monkeys was monitored daily. Six days after the onset of menses, daily blood samples were collected by saphenous venipuncture. Serum was separated and stored at –20 C until assayed for estradiol and P concentrations, as previously described (5). The first day of low serum estradiol after the mid-cycle estradiol peak has been demonstrated to correspond with the day after the LH surge, and therefore was termed d 1 of the luteal phase (26).

CL (n = 3–4 per stage) were isolated and dissected from anesthetized monkeys during the early (luteal d 3–5), mid (luteal d 6–8), mid-late (luteal d 10–12), late (luteal d 14–16), and very-late (luteal d 17–18) luteal phase, according to the protocol previously described in our laboratory (27). A portion of the CL was frozen in liquid nitrogen and stored at –80 C for isolation of total RNA using TRIzol (Invitrogen, Carlsbad, CA). A second portion was fixed in formalin for immunohistochemical analysis of caspase-2 and -3 proteins. The remainder of the CL was used for protein isolation.

Hormone ablation/replacement treatment was administered daily in adult rhesus monkeys on d 9–11 of the luteal phase, as previously described (5). These days were selected because the CL begins to regress near the end of this time period; in addition, this is the window of CL rescue in the primate by CG in fertile cycles (1). To assess the potential regulatory role of LH and P, females were assigned randomly to one of five treatment groups (n = 4 per group): control (no treatment), antide [GnRH antagonist, 3 mg/kg, sc injection; previously demonstrated to suppress circulating bioactive LH levels and luteal function (28)], antide + recombinant human LH [LH; 40 IU three times a day, im injection; Serono Reproductive Biology Institute, Rockland, MA; previously shown to restore LH function (28)], antide + LH + trilostane [TRL; a 3ß-hydroxysteroid dehydrogenase inhibitor previously demonstrated to ablate luteal P production (27); 600 mg administered in an oral dose (8 ml of sucrose and Tang vehicle; Sanofi Research Division, Northumberland, UK)], and antide + LH + TRL + R5020 [R5020; a nonmetabolizable progestin, 2.5 mg administered as a 2.5-ml (total volume) sc injection in a vehicle of sesame oil; DuPont, Boston, MA; previously shown to restore progestin function (27)]. On d 12, the CL was removed from anesthetized monkeys during an aseptic ventral midline surgery (27), weighed, and portions were divided and either immediately frozen for subsequent RNA or protein isolation or processed for histology (27). Portions of these tissues were used in reported studies on the regulation of matrix metalloproteinases by LH and steroids in the monkey CL (5).

Analysis of caspase-2, -3, -8, and -9 mRNA

Total RNA was extracted from individual CL treated with 1 µg DNase (Invitrogen) and analyzed separately. RT was performed for 2 h at 37 C in a 20-µl reaction volume using Moloney murine leukemia virus reverse transcriptase (Invitrogen) as described previously (4). PCR was performed to analyze caspase-3, -8, and -9 cDNA in a 25-µl volume containing an empirically determined amount of the RT reaction, 1 µl of the 10 mM specific primer set based on human sequences (Table 1Go), 2.5 µl 10x strength Taq buffer (Clontech, Palo Alto, CA), 1 µl 10 mM deoxynucleotide triphosphates, 0.75 µl Advantage 2 Taq (Clontech). The reaction was initiated at 94 C for 1.5 min, followed by 94 C for 30 sec, 55 or 60 C (depending on the caspase; see Table 1Go) for 30 sec, and 72 C for 2 min for 30–35 cycles (see Table 1Go), and a final extension at 72 C for 5 min. Aliquots of the PCR products were electrophoresed through a 1.4% agarose gel stained with 0.1 µg/ml ethidium bromide. Cyclophilin cDNA was analyzed as an internal control as previously described (29). Gels were visualized on a UV transilluminator and photographed. The gel bands were quantified using Quantity One software (Bio-Rad, Hercules, CA). And the PCR products were purified for confirmative sequencing by the ONPRC Molecular and Cell Biology Core facility, using an ABI 3100 automated sequencer.


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  		TABLE 1. Sets of forward (FOR) and reverse (REV) primers designed for RT-PCR amplification of caspase mRNAs from the macaque corpus luteum

 
Because caspase-2 was not as readily detectable as the other caspases by the semiquantitative (sq) RT-PCR technique described above, real-time PCR was used for analysis. Using the same cDNA samples as above, PCR was performed using primers based on the human caspase-2 sequence. The resulting macaque PCR product was sequenced and used to design TaqMan primer and probe sets for the real-time assay (Table 2Go; Primer Express software; PerkinElmer Applied Biosystems, Foster City, CA) as previously described (30). Probes were labeled with the 5' reporter dye 6-carboxyfluorescein and the 3' quencher dye 6-carboxytetramethylrhodamine. To determine optimal concentrations of assay components, a matrix of varying primer concentrations was used. As an internal control for the amount of total RNA added to each RT reaction and to normalize the target signal, 18S RNA was used as an active endogenous control in each well. Real-time PCR was performed in a 10-µl final volume containing: 250 nmol/liter TaqMan caspase-2 probe (labeled with the 5' reporter dye VIC), 300 nmol caspase-2 forward and reverse primers, 250 nmol/liter TaqMan 18S probe (labeled with the 5' reporter dye VIC), 40 nmol/liter forward and reverse 18S primers, 20 ng equivalent RNA, and 5 µl TaqMan Universal PCR master mix containing ROX dye as a passive reference (PE Biosystems). The PCR was run in a sealed 96-well optical plates and the thermal cycler conditions were: 2 min at 50 C, 10 min at 95 C, and 40 cycles of 15 sec at 95 C and 1 min at 60 C (primer annealing/extension). During the amplification cycles, the sequence detector (ABI PRISM 7700 Sequence Detection System, PE Applied Biosystems) monitored real-time PCR amplification by quantitatively analyzing changes in fluorescence emissions in each well. The number of amplification cycles for the fluorescence to reach a determined threshold level (CT) was recorded for every unknown and an internal standard curve. The internal standard curve was generated from five 10-fold dilutions of pooled early CL samples and used for relative mRNA quantification. The CT values for unknown samples were used to extrapolate the amount of RNA equivalents from the internal standard curve. The RNA equivalent values were then divided by complimentary 18S RNA equivalent values derived from the same internal standard curve.


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  		TABLE 2. Sets of forward (FOR) and reverse (REV) primers and probe designed for real time PCR amplification of caspase-2 mRNA from the macaque corpus luteum

 
Analysis of caspase-2 and -3 protein

Portions of the CL were fixed in 10% neutral buffered formalin (Richard-Allen Scientific, Kalamazoo, MI) for 1 wk. Tissue was then dehydrated in a series of ethanol solutions (50, 70, and 100%) and paraffin embedded. For immunohistochemistry, 6-µm sections were deparaffinized and hydrated through xylenes and a graded series of ethanol, as reported previously (31). Sections were incubated in PBS before pressure cooker-antigen retrieval in citrate buffer (Citra; BioGenex Laboratories, Inc., San Ramon, CA). Endogenous peroxidases were then quenched with a 10-min incubation in 3% H2O2. Sections were placed in a blocking buffer (1.5% normal horse serum in PBS), then incubated with primary antibody in normal horse serum PBS buffer for 1 h at room temperature and overnight at 4 C. Concentrations for human caspase-2, -3, -8, -9 (sc-626, sc-1226, sc-7890, sc-7885; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies were 1:200 (-2), 1:100 (-3), and 1:50 (-8 and -9) respectively. According to the manufacturer, these antibodies recognize both the inactive as well as the active form of the caspase. Primary antibody was detected using a biotinylated antirabbit IgG secondary antibody (1:1000; Vector Laboratories, Burlingame, CA) and the Vector ABC-Elite Kit, visualized with nickel-enhanced Sigma Fast diaminobenzidine substrate (Sigma, St Louis, MO) and counter-stained with hematoxylin. For each caspase examined, negative controls lacking primary antibody were processed on adjacent tissue sections.

Concentrations of the active form of caspase-3 in homogenized CL from different stages of the luteal phase were quantitated by ELISA (Quantikine-human active caspase-3, KM300; R&D Systems Inc., Minneapolis, MN). This ELISA measures the relative amount of caspase-3 large subunit modified with biotin-ZVD-fluoromethylketone. Because the modification requires that the large subunit is present in an active caspase-3, the amount of active caspase-3 is directly proportional to the amount of biotin-ZVKD- fluoromethylketone-modified large subunit. Based on the manufacturer’s instructions, 5 µg of the extracted proteins from the CL were used in the assay. The levels of the active protein were determined by absorbance, at 450 nm, against a standard curve. Each sample was tested in duplicate and negative controls (blanks, without sample) were subtracted.

Caspase-2, -3, -8, and -9 activity assays

A fluorometric assay kit (630225; Clontech), which contains fluorogenic substrates specific for different caspases (2, 3, 8, and 9) immobilized in the wells, was used to evaluate enzyme activity in the primate CL. Ten micrograms of the extracted proteins in homogenization buffer (50 mM Tris HCL, 150 mM NaCl, 10% glycerin, and 1% Triton X-100) containing protease inhibitors (Halt Protease Inhibitor Cocktail Kit, EDTA free; Pierce Biotechnology, Rockford, IL) were added to the wells. The plate was incubated in the fluorescence plate reader at 37 C for 3 h, and fluorescence was read every 10 min. The activity was determined by fluorometric detection (excitation, 380 nm; emission, 460 nm) and the negative control (blank, without sample) was subtracted from all the samples. Results at 2 h were selected, as the manufacturer suggested. Baseline values of negative controls and samples with specific inhibitors did not increase during the 2-h interval.

Terminal deoxynucleotidyl transferase nick end labeling (TUNEL)

Nuclear DNA fragmentation in cells of CL from the different stages of the luteal phase was detected using the DeadEnd Colorimetric TUNEL System (G7130; Promega, Madison, WI), following the manufacturer’s instructions with minor modifications. Tissue sections were deparaffinized and rehydrated as described earlier for immunohistochemical analysis of caspase proteins. Also, the interval used to block endogenous peroxidase activity with 3% H2O2 was increased from 5 to 15 min. Finally, the sections were counterstained with hematoxylin.

Statistical analysis

Differences among experimental groups were analyzed using one-factor ANOVA followed by Student-Newman-Keuls method or Dunn’s test using the SigmaStat software package (SPSS, Chicago, IL). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analyses of caspase-2, -3, -8, and -9 mRNAs

Caspase mRNAs were detectable by sqRT-PCR (caspase-3, -8, and -9) or real-time PCR (caspase-2) in the macaque CL during the menstrual cycle. The sequences of the macaque cDNA products showed a homology to the corresponding human sequences of 97% (caspase-2), 94% (caspase-3), 93% (caspase-8), and 95% (caspase-9). Nevertheless, the PCR analyses revealed no significant changes in mRNA levels for caspase-2 (not shown), -3 (Fig. 1AGo), -8 (Fig. 1BGo), and -9 (not shown) in the macaque CL throughout its lifespan in the menstrual cycle. As defined earlier (30), the luteal lifespan during the spontaneous menstrual cycle in rhesus monkeys includes CL formation (early luteal phase, ECL), peak CL function (mid luteal phase, MCL), CL on the verge of regression (mid-late luteal phase, MLCL), the regressing CL (late luteal phase, LCL) and the regressed CL at menstruation (very-late luteal phase, VLCL).



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  		FIG. 1. sqRT-PCR results (mean ± SEM) for caspase-3 (A) and -8 (B) mRNA in the macaque CL during the early (ECL, d 3–5), mid (MCL, d 7–8), mid-late (MLCL, d 10–12), late (LCL, d 14–16), and very-late (VLCL, d 17–19) luteal phase. Values were standardized to cyclophilin (mRNA) control values (n = 3–4 per group). There were no significant differences in mRNA levels for either caspase in the CL throughout its lifespan in the menstrual cycle (P > 0.05).

 
Analysis of caspase-2 and -3 protein

Caspase-2 and -3 immunohistochemistry. Immunohistochemical staining for caspase-2 (Fig. 2Go) was detectable in the CL during the early, mid, and mid-late luteal phase (Fig. 2Go, A–C), but appeared to increase at the late (Fig. 2DGo) and peak at the very-late stage (Fig. 2EGo). The negative control without the primary antibody showed no staining (Fig. 2FGo). Immunolabeling for caspase-2 was primarily found in the cytoplasm of granulosa luteal cells (g), but not in theca luteal cells (not shown), nor the surrounding stroma (s). By the mid-late luteal phase, some cells displayed no staining whereas others were stained intensely. At the late to very-late phase, many cells displayed intense staining.



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  		FIG. 2. Immunohistochemistry for caspase-2 in the macaque CL during different stages of the luteal phase in the menstrual cycle. Immunoreactivity was low in the CL during the early (A) and mid (B) luteal phase, more evident in a few cells especially by mid-late (C) luteal phase, but most pronounced at the late (D) and very-late (E) luteal phase. Staining was evident in the cytoplasm of granulosa luteal cells (g), but not in the theca luteal cells, nor in the stroma (s). The negative control incubated without the primary antibody displayed no staining (F).

 
Specific staining for caspase-3 (Fig. 3Go) was also found in the CL at all stages of the luteal phase compared with controls (Fig. 3FGo), appearing most intense at the mid-to-late stages (Fig. 3Go, B–D). Immunoreactivity for caspase-3 was primarily found in the granulosa-luteal cells (g) and theca-luteal cells (t), but not in the stroma (s). Notably, by the mid-late stage (Fig. 3CGo), caspase-3 staining appeared localized to the nucleus as well as the cytoplasm.



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  		FIG. 3. Immunohistochemistry for caspase-3 in the macaque CL during specific stages of the luteal phase in the natural menstrual cycle. Immunostaining was found in the granulosa luteal cells (g) and in the theca luteal cells (t), but not in the stroma (s) nor in the negative control (F). Staining during the early (A) and mid (B) luteal phase was cytoplasmic whereas in the mid-late (C), late (D), and very-late (E) luteal phase staining was also found at the nucleus (arrows). Specific staining for caspase-3 was detectable throughout the luteal phase but appeared most intense by the mid-to-late stages.

 
Immunoassay of active caspase-3. Low levels of the active form of caspase-3 (Fig. 4Go) were detected in the CL during the early-to-mid luteal phase. However, levels increased and peaked (P < 0.05) at the mid-late stage, followed by a decrease at subsequent stages. Active caspase-3 protein levels were 58-fold higher in mid-late stage compared with the preceding midluteal phase.



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  		FIG. 4. Levels of active caspase-3 protein as measured by immunoassay in the macaque CL during the early (ECL, d 3–5), mid (MCL, d 7–8), mid-late (MLCL, d 10–12), late (LCL, d 14–16), and very-late (VLCL, d 17–19) luteal phase. Groups with different letters over the error bars (a, b, c) represent significant differences (P < 0.05) between stages of the luteal phase (n = 3–4 per group).

 
Caspase activity assays

The activities of caspase-2 (not shown), -3 (Fig. 5AGo), -8 (Fig. 5BGo), and -9 (not shown) displayed a similar pattern during the CL lifespan. Activity was low in early luteal phase, increased (P < 0.05) to peak levels at the mid-late stage and tended to decline by the late and very-late stage. Assessment of caspase-2 revealed a 5-fold increase (P < 0.05) between early and mid-late stage. Likewise, caspase-3, -8, and -9 activity increased (P < 0.05) 7-, 10-, and 5-fold, respectively by mid-late luteal stage. However, the decline from the mid-late to very-late stage was significant (P < 0.05) for caspase-3, but not for caspase-2, -8, or -9.



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  		FIG. 5. Enzyme activity (mean ± SEM) for caspase-3 (A) and -8 (B) in the macaque CL at specific stages during the luteal phase. Groups with different letters over the error bars (a, b) represent a significant difference (P < 0.05) in the activity between mid-late and other stages (n = 3–4 per group).

 
TUNEL assays

Nuclear DNA fragmentation was detected by TUNEL staining in the macaque CL throughout the luteal phase (Fig. 6Go). Interestingly, staining appeared to increase as the luteal phase progressed from the early (Fig. 6AGo) to very-late (Fig. 6EGo) stages. Specific staining was detected in granulosa-luteal (g) and theca-luteal (not shown) cells, as well as in some endothelial cells (e). Staining was not found in the negative control without the TdT enzyme (Fig. 6FGo), nor in the surrounding stroma (not shown).



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  		FIG. 6. In situ 3' end labeling (TUNEL) of macaque CL obtained at different stages of the luteal phase (ECL, A; MCL, B; MLCL, C; LCL, D; VLCL, E) of the menstrual cycle. Staining was found in some granulosa luteal (g; adjacent to positive cells) and theca luteal (not shown) cells and in some endothelial cells (e; adjacent to positive cells). As the luteal phase progressed, staining appeared to increase, resulting in a higher number of apoptotic cells in the VLCL (E). Negative control without the TdT enzyme (F); the granulosa luteal (g) and other cells in the CL, plus the surrounding stroma (s) showed no staining.

 
Effect of hormone ablation and replacements on caspase activity

Although in vivo antide treatment alone for 3 d did not significantly affect caspase activity compared with controls, antide + LH increased (P < 0.05) caspase-2 (not shown), -8 (Fig. 7BGo), and -9 activity (not shown). However, antide + LH treatment did not alter caspase-3 activity (Fig. 7AGo) compared with controls or antide alone. Addition of TRL to the antide + LH treatment significantly (P < 0.05) reduced caspase-2, -8 (Fig. 7BGo), and -9 activity to levels comparable to controls. Replacement with the progestin R5020 tended to increase caspase activity but this effect was not statistically significant. However, neither TRL nor R5020 treatment altered caspase-3 activity (Fig. 7AGo).



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  		FIG. 7. Enzyme activity (mean ± SEM) for caspase-3 (A) and -8 (B) in the macaque CL after different treatments during the mid-late (d 9–11) luteal phase. (Control; Antide (A); A + LH; A + LH + TRL; A + LH + TRL + R5020). Groups with different letters over the error bars (a, b) represent a significant difference (P < 0.05) in the activity between treatments (n = 3–4 per group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides the first evidence on the dynamics and possible regulation of multiple (both initiator and effector) caspases in the primate CL during the natural menstrual cycle. Employing ovarian tissues collected at specific intervals during the luteal phase, the data indicate that caspase-2, -3, -8, and -9 are expressed in the macaque CL throughout the luteal lifespan from early development to structural regression. However, the enzymatic activity of these caspases peaked at the onset of CL regression (mid-late luteal phase), and indeed declined as luteolysis progressed in the late luteal phase. Unexpectedly, LH replacement in GnRH antagonist-treated monkeys increased the activity of initiator (-2, -8, and -9) caspases, whereas steroid ablation (using the 3ß-hydroxysteroid dehydrogenase inhibitor, TRL) reversed the LH effects. The results are consistent with a role for caspases in the primate CL in the initial stages of luteal regression, with perhaps additional actions at other stages of the luteal lifespan. Also, luteotrophic gonadotropins (LH, CG) may regulate caspase protein/activity levels independent of transcription, but these effects could be indirect via locally produced steroids.

PCR analyses combined with sequence verification of the partial cDNAs, protein immunolabeling procedures, and enzyme activity assays confirmed the expression of three initiator (-2, -8, and -9) and one major effector (-3) caspase in the rhesus monkey CL at all stages of the luteal phase in spontaneous menstrual cycles. However, the mRNA levels for these four caspases did not vary significantly between stages of the luteal phase, i.e. from luteal development after ovulation through luteal regression at menstruation. Limited evidence suggests that caspase mRNA levels increase in the CL of domestic animals [e.g. -1 (32) and -3 (20)] after pharmacological PGF2{alpha} treatment to initiate luteolysis, but it is less clear that this occurs during the natural ovarian cycle (20, 33). We cannot rule out that luteal caspase expression increases acutely within the 3-d sampling intervals, which could be masked by other tissues in the luteal stages. However, the current results suggest that regulation of caspases in the primate CL during its natural lifespan in the menstrual cycle is not at the transcriptional level, but rather at protein translation or enzyme activation.

Attempts to localize caspases by immunohistochemistry to specific cell types in the CL were necessarily limited to caspase-2 and -3. Antibodies to human caspase-8 and -9 that were suitable for paraffin sections did not yield satisfactory results with macaque tissue. According to the manufacturer, the anti-caspase-2 and -3 antibodies recognize both the inactive and active forms of the caspases. Data in the literature (34, 35) support this premise for the caspase-3 antibody. But efforts to validate the caspase-2 antibody in our laboratory only detected the inactive form of caspase-2 by Western blotting under various conditions (data not shown). Notably, specific immunolabeling for caspase-3 was localized to the granulosa- and theca-luteal cells, whereas caspase-2 staining was evident in granulosa-luteal but not in theca-luteal cells. Adjacent stromal tissue, which did not exhibit specific staining, proved to be a valuable in situ control. These data suggest that the steroidogenic luteal cells derived from the granulosa and theca layers of the antecedent follicle express common effector caspases (i.e. caspase-3), but may differ in the role of initiator caspase (e.g. caspase-2) pathways. The evidence is consistent with limited reports of caspase-3 immunostaining in luteal cells of the human CL by midluteal phase (36, 37), plus healthy as well as regressing CL from nonprimate species (19, 24).

Interestingly, caspase-3 immunostaining was evident in the cytoplasm of luteal cells at all stages, but only became associated with the nucleus by the mid-late luteal phase. The inactive forms of caspases are found in various intracellular sites, but after activation the effector caspases in particular are translocated to specific compartments such as the nucleus (8). Thus, the association with the nucleus may reflect an increase in caspase-3 activity at mid-late luteal phase, which is consistent with the results of the enzyme assay. The divergence between nuclear localization of caspase-3, but not caspase-2, may relate to differences in the specificity of their respective antibodies for the active vs. inactive caspase, and/or to the sites of action of the effector vs. initiator caspase. Although not quantitative, the immunohistochemical data suggested that caspase-2 staining increased, both in numbers of positive cells and staining intensity, in the CL by the late luteal phase. In contrast, caspase-3 staining was appreciable in many cells by the midluteal phase. Thus, there may be posttransciptional control of caspase protein levels in the macaque CL. However, the remarkable increase in levels of active caspase-3 protein, as well as enzyme activity, in CL between the mid and mid-late luteal phase, strongly suggests that the primary regulation of caspases occurs at the posttranslational level.

The current data unequivocally indicate that the level of active protein (caspase-3) and enzyme activity (caspase-2, -3, -8, and -9) increases transiently in macaque CL at mid-late luteal phase. At this stage (d 10–12 post-LH surge), the CL is on the verge or just beginning luteal regression, as circulating P levels are still above baseline (>1 ng/ml) and CL weight has not declined from that at midluteal phase (30). These findings suggest that caspase activation is an early event near the onset of luteal regression, and may be important in functional as well as structural luteolysis in primates. Because caspase (especially the effector caspase-3) activation is considered a key event in apoptosis (8), the presence of active caspase-2, -3, -8, and -9 in mid-late to very-late luteal phase suggests that apoptosis occurs during luteolysis in the menstrual cycle of rhesus monkeys. Although DNA-fragmentation-based approaches, such as TUNEL, have their limitations (38), investigators reported that TUNEL staining correlates with other morphological and biochemical (e.g. DNA laddering on agarose gel electrophoresis) indices of apoptosis in the CL, including human luteal tissue (39, 40). In the current study, TUNEL staining was evident in the macaque CL throughout the luteal phase, but the numbers of positive-stained cells appeared to increase as the luteal lifespan progressed. Thus, it is possible that the low level of caspase activity in the early-to-mid luteal phase is associated with a few cells undergoing apoptosis (21), and that increased caspase activity depicts an incremental increase in cell death (steroidogenic and endothelial cells) as luteolysis begins in earnest. Other reports (e.g. Ref. 39) of initial DNA cleavage and TUNEL labeling of luteal cells in the human CL at midluteal phase would suggest that apoptosis is not limited to the final structural regression of the CL.

Nevertheless, the role of various initiator (-2, -8, and -9) and effector (-3) caspases in controlling luteal structure-function requires further study. Rueda and colleagues (24) performed in vivo and in vitro studies in caspase-3 null (–/–) mice which suggested that this enzyme was essential for the structural, but not functional, regression of the CL. Also, inhibitors of caspase-1 and -3 homologs suppress an apoptotic phenotype in cultured CL from rabbits (41). Alternatively, Kato and colleagues (33) suggested that changes in caspase-3 activity in the rat CL of pregnancy were not consistent with the incidence of apoptotic cells, and suggested that CL regression after pregnancy is associated with a caspase-3-independent mechanism of apoptosis. Perhaps caspase-initiated apoptosis is of greater significance in species with abrupt luteal dissolution (e.g. the hamster (42)) than those with a more gradual disappearance of the CL, including primates. Likewise, one must consider the possibility, emerging from other experimental systems, that caspases can have other functions in cells to control proliferation, differentiation, or cytokine production (43, 44). The latter action in CL at mid-late luteal phase could be influential in the onset or progression of luteolysis.

The evidence that GnRH antagonist treatment, which eliminates luteotropic support by suppressing circulating LH levels (28), did not alter caspase activity—and that LH replacement increased the activity of initiator (-2, -8, and -9) caspases—was counter to our working hypothesis. We previously used this protocol (27), and these same tissues (5, 45), to identify LH-regulated gene products in the developed CL in rhesus monkeys. Based on the evidence of elevated caspase activity during the initiation of luteolysis in the natural cycle, we suspected the removal of luteotropic support would elicit at least in part, similar pathways leading to loss of luteal structure-function. The lack of effect of GnRH antagonist exposure could be due to failure to sample CL at the appropriate time instead of after 3 d of exposure. Alternatively, the data may support the premise championed by Fraser et al. (46) that the mechanisms and forms of luteal degeneration associated with natural luteolysis during the menstrual cycles are different from those associated with pharmacological luteolysis associated with GnRH antagonist or prostaglandin treatment. Nevertheless, removal of gonadotropin support increases caspase-2 and -3 expression in monkey preovulatory follicles (47), and one might expect that maintenance of luteotropic support in GnRH-antagonist-treated monkeys would suppress caspase activity in the CL (48). However, there are published reports that addition of tropic hormones (i.e. LH-CG (49, 50) and perhaps their intracellular mediator, cAMP (49), can induce effector (-3 and -7) caspases in granulosa cells of preovulatory follicles, in association with decreased apoptosis (50). The mechanisms and importance of gonadotropin-stimulated caspase activity in the preovulatory follicle and CL awaits investigation.

The ability of a 3ß-hydroxysteroid dehydrogenase inhibitor, TRL, to suppress the increase in caspase activity elicited by LH treatment, suggests that this effect is mediated by locally produced steroids. We previously used this approach of steroid ablation and progestin (R5020) replacement to distinguish between LH-regulated processes in the macaque CL that are independent vs. dependent or mediated at least in part by local P action (5, 27, 45). In the current study, progestin replacement had little effect to prevent suppression of caspase activity in the developed CL by TRL. Previous reports have suggested that P suppresses caspase-3 activity (51, 52), cellular apoptosis (53) and luteal degeneration (5, 54) in CL of species with long luteal phases during the ovarian cycle, e.g. domestic animals and primates. Because steroid ablation reduced caspase activity, it is difficult to consider a suppressive action of P in this regimen. Rather, the evidence suggests that other nonprogestin steroids are mediating LH actions to increase caspase activity. Because the macaque CL reportedly contains both estrogen receptor-ß (27) and androgen receptor (26) these steroids warrant further experiment. Nevertheless, the current data suggest that gonadotropic hormones and local steroids act primarily to regulate initiator (-2, -8, and -9) caspase activity rather than the main effector caspase-3, in the primate CL.

In conclusion, our findings indicate that several members of the caspase (-2, -3, -8, and -9) family are expressed and active in the rhesus monkey CL throughout the luteal phase of the natural menstrual cycle. The transient rise in initiator and effector caspase activity at mid-late luteal phase suggests that this system is important during the initiation and early stages of luteal regression; however, its role in apoptosis or other cellular processes during the luteal lifespan awaits further investigation. Regulation of these caspases in the primate CL was not at the transcriptional level, but rather at the level of translation and/or enzyme activation. The primary luteotropic hormone of CL, LH, can enhance the activity of effector (-2, -8, and -9) caspases after 3-d exposure, but much of this effect may be mediated by locally produced steroids other than P.


   Acknowledgments
 
We are grateful for the expert contributions of the animal care staff and surgical unit of the Division of Animal Resources, the outstanding technical support of the Endocrine Services, Imaging & Morphology, and Molecular & Cell Biology Core Laboratories at ONPRC. Recombinant human LH (Serono Reproductive Biology Institute) and TRL (Sanofi Pharmaceutical Inc., Grand Valley, PA) were generously donated for this project. A special thanks to Dr. Jon Hennebold, his laboratory associates, and Dr Alejandro Lomniczi for their scientific advice and assistance in the TUNEL assay and other techniques, and to Dr. Ted Molskness and Ms. Carol Gibbins for assistance in preparing this manuscript.


   Footnotes
 
This research was supported by National Institutes of Health, National Institute of Child Health and Human Development (NICHD) Grants HD20869 (to R.L.S.) through a cooperative agreement and U54 HD18185 as part of the Specialized Cooperative Centers Program in Reproduction Research. Support was also provided by NICHD/Fogarty D43 TW00668 (to M.C.P.), National Research Service Award HD042896 (to K.A.Y.), and National Center for Research Resources RR00163 (to R.L.S.).

Present address for M.C.P.: Instituto de Biologia y Medicina Experimental, Vuelta de Obligado 2490, C1428 ADN, Buenos Aires, Argentina.

Present address for K.A.Y.: Department of Biological Sciences, California State University-Long Beach, 1250 Bellflower Boulevard, Long Beach, CA 90840-3702.

First Published Online January 25, 2005

Abbreviations: CG, Chorionic gonadotropin; CL, corpus luteum; P, progesterone; sq, semiquantitative; TRL, trilostane; TUNEL, terminal deoxynucleotidyl transferase nick end labeling.

Received November 11, 2004.

Accepted January 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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