Reproduction   citetrack
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS  
Reproduction (2006) 131 103-111
DOI: 10.1530/rep.1.00751
Copyright © 2006 Society for Reproduction and Fertility
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goyeneche, A. A
Right arrow Articles by Telleria, C. M
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goyeneche, A. A
Right arrow Articles by Telleria, C. M

RESEARCH

Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis

Alicia A Goyeneche, Jacquelyn M Harmon and Carlos M Telleria

Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, South Dakota 57069, USA

Correspondence should be addressed to C M Telleria; Email: carlos.telleria{at}usd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The corpus luteum is a transient endocrine gland specializing in the production of progesterone. The regression of the corpus luteum involves an abrupt decline in its capacity for producing progesterone followed by its structural involution, which is associated with apoptosis of the luteal cells. An in vitro experimental approach is needed to study the molecular mechanisms underlying hormonal regulation of luteal cell death under defined experimental conditions. In this study, we investigated simian virus-40-transformed luteal cells to determine whether they can be driven to apoptosis and, if so, to define the intracellular pathway involved. Luteal cells were cultured in the presence or absence of fetal bovine serum for 24 or 48 h. Under serum starvation conditions, the luteal cells underwent growth arrest accompanied by cell death as evaluated by dye exclusion, and confirmed by two-color fluorescence cell viability/cytotoxicity assay. We next studied whether serum starvation-induced death of luteal cells occurred by apoptosis. Morphologic features of apoptosis were observed in cells stained with hematoxylin after being subjected to serum starvation for 48 h. The apoptotic nature was further confirmed by in situ 3'-end labeling and fragmentation of genomic DNA. Apoptosis of serum-deprived luteal cells was dependent upon caspase activation. Serum starvation induced cleavage of poly (ADP-ribose) polymerase (PARP), suggesting that caspase-3 had been activated under the stress of withdrawal of growth factors. This was confirmed by cleavage of full-length procaspase-3. Finally, the fact that serum starvation promoted the cleavage of full-length procaspase-9 and the decrease in the expression of endogenous Bid, a BH-3-only proapoptotic protein of the Bcl-2 family, indicates that the intrinsic (i.e., mitochondrial) pathway of apoptosis was activated. In summary, we have characterized an in vitro experimental model of luteal cell death that can be utilized to evaluate the role of hormones in apoptosis of luteal cells under defined culture conditions, and to study the mechanism of luteal regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Luteal regression is a two-phase process, the first of which is known as functional and is associated with the loss in the capacity of the luteal cells to produce progesterone. The second phase, or structural, occurs well after the initial decline in progesterone output and is associated with programmed death of the luteal cells (for review, see Davis & Rueda (2002) and references therein). Programmed cell death in the corpus luteum follows a pattern of apoptosis characterized by initial condensation of the nuclear chromatin followed or accompanied by nucleosomal fragmentation of DNA and formation of apoptotic bodies, which eventually are eliminated by phagocytosis. Morphologic and biochemical features of apoptosis have been observed in regressing corpora lutea of various mammalian species including pigs (Bacci et al. 1996), sheep (Rueda et al. 1995), cattle (Juengel et al. 1993, Rueda et al. 1997), man (Shikone et al. 1996), hamsters (McCormack et al. 1998) and rats (Bowen et al. 1996, Matsuyama et al. 1996, Gaytan et al. 1998, 2000, Telleria et al. 2001). Hence, programmed cell death and apoptosis are frequently used as equivalent descriptors of luteal cell death.

The number of apoptotic bodies that can be seen at a given time within a corpus luteum is relatively low (Bowen & Keyes 2000, Gaytan et al. 2001, Telleria et al. 2001, Goyeneche et al. 2002, 2003a, 2003b). This is in contrast to what happens in the ovarian follicle undergoing atresia, within which a large number of granulosa cells display morphologic features of apoptosis simultaneously (Byskov 1974, Hughes & Gorospe 1991, Tilly et al. 1991, Tilly 1994). The low number of apoptotic cells within the regressing corpus luteum could be the consequence of a low rate of apoptosis taking place at the same time, or of a rapid clearance of the apoptotic bodies by phagocytosis. Whichever the case, the low number of apoptotic figures observed in vivo at any given time has made it difficult to study apoptosis during luteal regression. Consequently, experimental approaches to study luteal apoptosis are needed to understand its mechanisms of control and hormonal regulation. Efforts have been made in this respect by generating an ex vivo model whereby corpora lutea incubated under serum-free conditions undergo extensive apoptosis, which is proportional to the time of incubation. By placing the corpus luteum ex vivo, the tropic support that it had in vivo is removed, allowing the apoptotic machinery to become active. This experimental approach was first developed with rabbit corpora lutea (Dharmarajan et al. 1999) and adapted for rat corpora lutea (Goyeneche et al. 2002, 2003a, 2003b, Abdo et al. 2003). The approach has the advantage of synchronizing cell death along the gland, permitting study of a large number of cells undergoing apoptosis at the same time. This ex vivo model was recently used to demonstrate that the degree of apoptosis that the gland undergoes once removed from the organism depends upon changes in the in vivo hormonal environment to which the gland was exposed. Elevation in circulating concentrations of prolactin, progesterone or androstenedione was reflected in prolonged times needed for cell death to occur in culture (Goyeneche et al. 2002, 2003a, 2003b), whereas administration of estrogen accelerated DNA fragmentation (Goyeneche & Telleria 2005). Thus, it is clear that the ex vivo model of luteal apoptosis allowed specific questions regarding the role of hormones in luteal cell death to be answered. Yet, in order to clarify the molecular mechanisms of hormonal control of luteal cell survival and cell death, an in vitro model of a homogeneous population of luteal cells is needed. In this work, we provide evidence of the characterization of an in vitro experimental model of luteal cell death by subjecting simian virus-40-transformed rat luteal cells to serum starvation in the presence of defined culture conditions. We demonstrate that transformed luteal cells display features of apoptosis in vitro associated with the activation of the mitochondrial pathway of apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Luteal cell line
The simian virus 40-transformed rat luteal cell line termed GG-CL was used (Sugino et al. 1998). Culture media consisted of RPMI 1640 (Mediatech, Herndon, VA, USA) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Laurenceville, GA, USA), 10 mM HEPES (Mediatech), 4 mM L-glutamine (Mediatech), 0.45% D-(+)glucose (Sigma), 1 mM sodium pyruvate (Mediatech), 1 x nonessential amino acids (Mediatech), 100 IU penicillin (Mediatech) and 100 µg/ml streptomycin (Mediatech). Cells were cultured at 37 °C in a humidified atmosphere in the presence of 5% CO2 to 60% confluence, and then subjected to conditions of serum starvation for various times. Control and starved cells were cultured in the same environment with identical media except for the lack of FBS for the starved cells.

Evaluation of cell growth, cell viability and cell death
To evaluate cell growth, cells were seeded at a density of 50 000 cells per well in six-well plates. The cells were cultured in the presence of serum for an additional 48 h, and either maintained in the presence of serum (+FBS) or in starvation media (–FBS) for 48 h. Cells were trypsinized and counted with a hemocytometer and the trypan blue exclusion method after 0, 24 and 48 h of incubation. The total number of living and dead cells was recorded for each experimental group in triplicates. The experiment was repeated three times.

For further analysis of viability and death, cells were cultured on sterile eight-well chamber slides at a concentration of 10 000 cells per well and subjected or not to starvation conditions. At the end of the incubation, cells were analyzed by a live/dead viability/cytotoxicity assay according to the instructions of the manufacturer (Molecular Probes, Eugene, OR, USA). In brief, cells were washed with PBS, and incubated for 45 min at room temperature in the presence of appropriate concentrations of cell-permeant calcein AM, which is a substrate for ubiquitous intracellular esterases, and ethidium homo-dimer 1 (EthD-1), which enters the cells having damaged plasma membranes and stains the DNA. At the end of the incubation, excess solutions were removed, the slides were mounted, and the labeled cells were observed with a Leica DM/LB microscope (Leica Microsystems, Plymouth, MN, USA). Calcein formed from calcein AM is well retained within live cells, producing an intense uniform green fluorescence. EthD-1 undergoes enhancement of fluorescence upon binding to DNA, producing a bright red fluorescence in dead cells, but it is excluded by the intact plasma membrane of live cells.

In situ detection of apoptosis
To evaluate apoptosis, luteal cells were cultured in eight-well chamber slides. Cells were grown in media with or without FBS for 48 h. At the end of the incubation, the cells were fixed with 4% paraformaldehyde (Sigma) and stained with hematoxylin (Sigma). Apoptotic cells were distinguished under an optic microscope by morphologic criteria by the procedure described by Van der Shepop et al.(1996) with slight modifications (Telleria et al. 2001). Apoptotic cells were identified as containing small, densely stained nuclei, nuclei with marginated chromatin, and multiple smaller nuclear fragments.

For further identification of apoptosis, cells were permeabilized in PBS containing 0.2% Triton X-100 (Sigma) for 5 min at room temperature and subjected to in situ 3' end labeling of the fragmented DNA by the DeathEnd colorimetric apoptosis system (Promega). This system end labels the fragmented DNA of apoptotic cells, using a modified terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay as described previously (Telleria et al. 2001).

DNA fragmentation
Progression of apoptosis over the time span was evaluated by analyzing internucleosomal cleavage of DNA. Floating and adherent cells were pelleted and digested overnight at 50 °C in a buffer composed of 100 mM NaCl, 10 mM Tris HCl (pH 8.0), 25 mM EDTA (pH 8.0), 0.5% SDS and 0.1 mg/ml proteinase K (Life Technologies, Rockville, MD, USA). The genomic DNA was extracted from the digested tissues with phenol/chloroform/isoamyl alcohol (25: 24: 1, v/v/v), precipitated, and digested for 60 min at 37 °C with 1 µg/ml ribonuclease (deoxyribonuclease-free; Roche). After extraction and precipitation, an equal amount of DNA for each sample (1 µg) was separated by electrophoresis on a 2% agarose gel, impregnated with SYBR Gold nucleic acid gel stain (Molecular Probes), examined using an ultraviolet transilluminator, and photographed with the Amersham Typhoon fluorescence imaging system. A 100-base pair (bp) DNA ladder (Promega) was used for determining the size of the DNA fragments.

Western analysis
Floating and adherent cells were pelleted, washed twice in PBS, and lysed by the addition of two volumes of RIPA buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM orthovanadate and 1 mM NaF. Cells were disrupted by passing them through a 21 gauge needle, and gently rocked on ice for 30 min. Lysates were centrifuged at 14 000 g at 4 °C for 15 min, and the supernatant was considered the whole-cell extract, which was assayed for protein content by using the bicinchoninic acid method (BCA; Pierce, Rockford, IL, USA). The whole-cell extract was appropriately diluted in 6 x concentrated electrophoresis sample buffer, boiled for 10 min, and stored at –80 °C until electrophoresed. Equivalent amounts of protein (25 µg) per point were separated by 12% SDS–PAGE gels and electroblotted onto PVDF membranes. The blots were blocked in 5% (v/v) nonfat milk in Tris–buffered saline (TBS) containing 0.1% (v/v) Tween 20 (T). Blots were then probed overnight with the appropriate dilution of each one of the primary antibodies. The membranes were washed 3 x 10 min in TBS-T and then incubated with a 1:25 000 dilution of peroxidase-conjugated secondary antibody for 30 min at room temperature. Blots were again washed 3 x 10 min in TBS-T and then developed by chemiluminescence and exposed to radiographic film. Blots were stripped and reprobed with an antibody directed against the ubiquitous protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to control for protein loading.

Antibodies for Western blot analysis
Primary antibodies for the following proteins were used at the designated dilutions: caspase-9 and caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA, USA); poly (ADP-ribose) polymerase (PARP) (1:2000, Cell Signaling); Bid (2 µg/ml; R&D Systems, Minneapolis, MN, USA); and GAPDH (1:4000; Abcam Inc., Cambridge, MA, USA). Secondary antibodies conjugated to horseradish peroxidase were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Cell growth arrest and cell death in serum-starved luteal cells
Transformed rat luteal cells show exponential growth as depicted by the number of viable cells observed when they were cultured in the presence of 10% of FBS (Fig. 1AGo, open circles). However, when a similar number of adherent cells were deprived of serum, they did not grow over the 48 h that the experiment lasted. Instead, the number of viable cells was similar to that recorded before the initiation of starvation (Fig. 1AGo, closed circles). Luteal cells that grew in the presence of serum had very low percentages of dead cells after 24- or 48-h incubation (Fig. 1BGo, open bars). However, there was a large increase in the number of dead cells after 24-h starvation, which was more evident after 48-h starvation (Fig. 1BGo, filled bars). Approximately 30% of the luteal cells still attached to the plate were capable of taking up the exclusion dye after 48-h starvation, indicating damage to their plasma membranes.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1 Evaluation of cell growth of luteal cells under serum-starvation conditions. Control and starved luteal cells were cultured in the same environment with identical media except for the lack of FBS for the starved cells. Cells were seeded at a density of 50 000 cells per well in six-well plates, allowed to grow for 48 h, and then subjected or not to starvation. Serum-starved (–FBS) and control (+FBS) cells were trypsinized and counted after 0, 24 and 48 h by the trypan blue exclusion method. The number of cells alive (A) and dead (B) was recorded for each experimental group in triplicates.

 
To confirm death in serum-starved luteal cells, we used two different fluorochromes that, combined, allow evaluation of the viability of the cells. Eth-D1 is a fluorochrome that enters cells with altered plasma membranes. Once inside the cells, Eth-D1 has a remarkable enhancement of fluorescence upon binding to DNA, producing a bright red fluorescence in dying cells. The accompanying fluorochrome, calcein AM, is a cell-permeant substrate of non-specific esterases found within the cytoplasm and nucleus of living cells. Nonfluorescent calcein AM is converted by esterase activity into a green fluorescence product in live cells. Both fluorochromes can be viewed simultaneously with a conventional fluorescein long-pass filter. Figure 2Go displays cells subjected to serum deprivation for 24 h (panels B and E) or 48 h (panels C and F) as compared with control cells cultured in the presence of serum (panels A and D). Results indicate an increase in cell death with time of starvation. Cells undergoing death have low esterase activity and high Eth-D1 binding (panels E and F). Panels A–C show the same preparations displayed from D through F, but observed with a bandpass filter that detect Eth-D1 fluorescence without detecting calcein fluorescence.



View larger version (49K):
[in this window]
[in a new window]
  		Figure 2 Evaluation of cell death in serum-starved luteal cells by a two-fluorochrome viability/cytotoxicity assay. Cells were cultured in eight-well chamber slides in the absence of serum for 24 h (B and E) or 48 h (C and F), and compared with cells cultured in the presence of serum (A and D). At the end of the experiment, the cells, without being fixed, were exposed to Eth-D1 and calcein AM. Panels A–C were obtained with a bandpass filter that recognizes the emission of Eth-D1 and marks cells with damaged plasma membrane that allow the fluorochrome to enter the cytoplasm and to bind DNA. Panels D–F were obtained with a longpass filter that recognizes both Eth-D1 and green fluorescence of calcein formed upon catalysis of calcein AM by intracellular esterases of living cells. Note that Eth-D1 was detected as red fluorescence when we used a bandpass filter, but as light red fluorescence when simultaneously detected with calcein with a longpass filter. x 400.

 
Cell death induced by serum starvation in luteal cells occurs by apoptosis
We next studied the nature of serum starvation-induced death in luteal cells by determining whether it follows an apoptotic process. In bright field, brilliant rounded cells could be observed in serum-starved cultures (Fig. 3DGo), but not in control cultures (Fig. 3AGo). This was accompanied by the presence of nuclei with condensed chromatin as stained with hematoxylin (Fig. 3EGo). Hardly any nuclei with condensed chromatin were observed in control cells (Fig. 3BGo). Finally, abundant nuclei displaying in situ 3'-end labeling of the DNA were observed in starved cultures (Fig. 3FGo), but not in control cultures (Fig. 3CGo). Bright round nuclei are characteristic of cells that are either dividing or beginning to detach from the culture plate. The former is not the case, as starved cells did not show features of mitosis (results not shown) and did not grow over the period of 48 h that the experiment lasted (Fig. 1AGo). The latter is most likely the case, as the presence of bright nuclei (Fig. 3DGo) coincided with features of apoptosis in cells stained with hematoxylin (Fig. 3EGo), as further confirmed by in situ 3'-end labeling of the DNA (Fig. 3FGo).



View larger version (94K):
[in this window]
[in a new window]
  		Figure 3 In situ detection of apoptosis in luteal cells cultured under serum starvation conditions. Luteal cells were cultured in eight-well chamber slides for 48 h with complete media (A–C) or with serum-starvation media (D–F). At the end of the incubation, the cells were observed with bright-field microscopy (A and D) or fixed with 4% paraformaldehyde and stained with either hematoxylin (B and E) or the DeathEnd colorimetric apoptotic system (a variant of TUNEL) (C and F). Panels A–C represent control luteal cells, whereas panels D–F represent serum-starved luteal cells. Arrows indicate apoptotic nuclei. x 400.

 
The apoptotic nature of the luteal cells cultured with starvation media was further confirmed by measuring fragmentation of the genomic DNA (Fig. 4Go). DNA isolated from luteal cells that had been starved for 24 or 48 h showed increasing degrees of fragmentation over the time of incubation when separated by electrophoresis. In contrast, cells that were cultured in the presence of serum did not show DNA fragmentation and had only low migrating genomic DNA. These results clearly confirm that serum-deprived luteal cells die by apoptosis.



View larger version (76K):
[in this window]
[in a new window]
  		Figure 4 Fragmentation of genomic DNA in serum-starved luteal cells. Genomic DNA was isolated from control cells cultured in the presence of serum (Co) or cells subjected to starvation conditions for 24 or 48 h. DNA was separated by electrophoresis on a 2% agarose gel, impregnated with SYBR Gold nuclei acid stain, examined using an ultraviolet transilluminator and photographed with the Amersham Typhoon fluorescence imaging system. A 100 base pair (bp) marker was run in parallel.

 
Apoptosis of serum-starved luteal cells associated with cleavage of caspase-9 and caspase-3, and decreased expression of endogenous Bid
To study the molecular mechanism involved in the apoptosis of the luteal cells subjected to starvation conditions, we cultured the cells in the presence or absence of FBS for 48 h, obtained whole-cell protein extracts from floating and adherent cells, and analyzed the expression of executer caspase-3. Caspase-3 usually drives most apoptotic mechanisms triggered by either the intrinsic (i.e., mitochondrial) or the extrinsic pathway of apoptosis. Cleavage of procaspase-3 was observed after serum starvation, giving rise to presumably active 17 kDa caspase-3 (Fig. 5AGo, upper panel). To determine whether caspase-3 was activated after serum starvation, the expression of PARP, a substrate for caspase-3 (Decker & Muller 2002), was analyzed. PARP precursor was cleaved to an 89 kDa product in serum-starved cells (Fig. 5AGo, middle panel), confirming caspase-3 activation.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 5 Cleavage of caspase-3 (A) and caspase-9 (B), and expression of endogenous Bid (C) in serum-starved luteal cells. Cleavage of caspase-3 and caspase-9, and abundance of endogenous Bid were studied by immunoblotting whole protein extracts obtained from luteal cells cultured with or without serum for 48 h, using specific antibodies. The activation of caspase-3 was confirmed by evaluating the cleavage of the caspase-3 substrate, PARP (middle panel in A). The housekeeping gene GAPDH was used as a loading control.

 
Because it has been reported that growth factor depletion generates a stress situation within the cells leading to the activation of the mitochondrial pathway of apoptosis (Gulbins et al. 2003, van Gurp et al. 2003), we tested this possibility in serum-deprived luteal cells. Luteal cells cultured in the presence of serum expressed the 51 kDa, full-length procaspase-9, whereas in serum-starvation conditions, the cells showed a decrease in the abundance of procaspase-9 that paralleled the expression of two cleaved products of 38 and 17 kDa respectively, the latter presumably being active caspase-9 (Fig. 5BGo). Finally, we could not detect caspase-8 expression in these luteal cells with a panel of various caspase-8 antibodies, yet we were able to detect the expression of Bid, a BH-3-only, proapoptotic member of the Bcl-2 family of proteins, which has been reported to be a target of caspase-8 and to amplify the apoptotic process (Esposti 2002, Degli Esposti et al. 2003).

The expression of Bid was abundant when the cells were cultured with serum but abruptly declined after 48-h serum starvation (Fig. 5CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
A growing body of evidence supports the association of luteal regression with an apoptotic phenomenon. However, the bulk of such studies evaluate the very latest steps of apoptosis, mostly related to activation of executioner caspase-3, fragmentation of DNA and formation of apoptotic bodies (for review, see Davis & Rueda 2002). The involvement of the mitochondria in luteal apoptosis has received less attention. In a recent study performed in mares, rarefaction and distortion of mitochondrial cristae were associated with loss of steroidogenesis in the corpus luteum and were considered an early maker of apoptosis (Al-Zi’abi et al. 2002). A closer involvement of the mitochondria in ovarian apoptosis was reported by Khan et al.(2000). Using human luteinized granulosa cells induced to apoptosis with the protein kinase inhibitor staurosporine, the authors showed mitochondrial cardiolipin levels to be reduced, and cleavage of procaspase-9 to presumably active caspase-9 to be associated with activation of caspase-3 and PARP cleavage. In luteal cells in which apoptosis was triggered by a gondadotropin-releasing hormone (GnRH) agonist, Papadopoulos et al.(1999) showed an association between fragmentation of DNA, decrease in mitochondrial levels of antiapoptotic protein Bcl-xL, and increase in mitochondrial levels of the proapoptotic protein Bax. The involvement of the mitochondrial pathway of apoptosis in the rat corpus luteum was also inferred from studies in which apoptosis of the luteal cells was induced by microgravity (Yang et al. 2002). Luteal cells subjected to microgravity lost the mitochondrial membrane potential and underwent apoptosis. Furthermore, mitochondrial dysfunction led to impaired steroidogenesis because progesterone production was dramatically inhibited. By using large rat luteal cells that had been transformed with the simian virus-40 (Sugino et al. 1998), we have confirmed that the mitochondrial pathway of apoptosis is operational. Luteal cells deprived of growth factors underwent cell death by apoptosis involving activation of the intrinsic apoptotic pathway as indicated by cleavage of initiator caspase-9. The fact that caspase-9 cleavage was associated with DNA fragmentation and cell death in serum-deprived luteal cells provides an experimental approach to investigate further the role of the mitochondria in luteal cell death.

The importance of caspase-3 as a mediator of apoptosis in luteal regression has been demonstrated in studies with caspase-3-null mice. The corpus luteum obtained from these animals showed attenuated rates of apoptosis and delay in the involution of the gland (Carambula et al. 2002). In serum-deprived luteal cells, we showed the activation of caspase-3 demonstrated by its cleavage and by the cleavage of its substrate, PARP, confirming the participation of this executer of apoptosis in luteal cell death. Therefore, because this in vitro model of luteal cell death mimics the activation of caspase-3 that takes place during regression of the corpus luteum in vivo, it can be used to clarify the molecular mechanism leading to caspase-3 activation in luteal cells.

Multiple studies demonstrate that the extrinsic pathway of apoptosis triggered by activation of death receptors is operational in luteal cells (Kuranaga et al. 1999, Abdo et al. 2003, Carambula et al. 2003). Under conditions of serum starvation, however, we could not detect activation of this pathway, yet we were able to show that endogenous Bid, a BH-3-only member of the Bcl-2 family of proteins (Yin 2000, Esposti 2002), was decreased in whole-protein extracts of serum-starved luteal cells. It is known that Bid can be cleaved by initiator caspases-2 (Guo et al. 2002) and -8 (Degli Esposti et al. 2003), and that truncated Bid (tBid) favors mitochondrial damage, and links the extrinsic and intrinsic apoptotic pathways (Esposti 2002). Thus, the cleavage of Bid, together with the activation of caspases-9 and -3 in serum-starved rat luteal cells, suggests that, in the presence of apoptogenic signals such as tumor necrosis factor alpha (TNF{alpha}) or Fas ligand (FasL), the scenario for physiologic cell death during luteal regression may involve the cooperation of both extrinsic and intrinsic apoptotic pathways.

The experimental model of luteal cell death characterized in this work can be useful for studying the intracellular mechanism leading to luteal cell death, and evaluating the role of hormones in luteal regression under defined culture conditions. This is because the GG-CL luteal cells express the glucocorticoid receptor and the estrogen receptor ß (Sugino et al. 1998) and respond to the action of glucocorticoid (Sugino et al. 1997), progestin (Sugino et al. 1997) and estrogen (Telleria et al. 1998).

An initial limitation in the use of these cells to study hormonal regulation of apoptosis is that, when compared with their primary counterparts, they lack the expression of receptors for key luteal regulators such as luteinizing hormone (LH) and prolactin (Sugino et al. 1998). However, such limitations can be overcome by forcing the expression of those receptor genes. In fact, we have recently used this experimental approach to study the effects of prolactin on luteal regression. We showed that the addition of prolactin to the culture medium significantly reduced the extent of DNA fragmentation induced by serum deprivation in luteal cells that had been permanently transfected with the long form of the prolactin receptor (Goyeneche et al. 2003b). These results allowed us to suggest that the survival effect of prolactin in luteal cells involves not only the stimulation of steroidogenesis, as is well known in rodents (Risk & Gibori 2001), but also the interference with apoptosis.

Another avenue that can be explored by the model of serum deprivation-induced death of immortalized luteal cells may be the understanding of the mechanism involved in the removal of residual bodies generated during death by apoptosis. There is ample evidence that macrophage invasion and phagocytosis of the apoptotic bodies takes place in regressing corpora lutea (Hehnke et al. 1994, Townson et al. 1996, Gaytan et al. 1997, Naftalin et al. 1997, Bowen et al. 1999). Thus, because primary cultures of rat macrophages are feasible (Sugino et al. 1996), and rat macrophage cell lines are available (Rao et al. 2002), it seems reasonable to perform experiments in which transformed luteal cells are induced to die by serum deprivation in the presence of autologous macrophages.

Even though apoptosis appears to be the most relevant mechanism of cell death during luteal regression, another mechanism of cell death that could be associated with apoptosis is autophagocytosis. This mechanism of cell death involves a process of autodegradation of cellular components by enclosing them in cytoplasmic vacuoles (Shintani & Klionsky 2004). Evidence of autophagocytosis in the corpus luteum arises from studies of cell death during luteal regression in the marmoset monkey (Young et al. 1997, Fraser et al. 1999). Morphometric studies conducted in corpus luteum in which regression was induced by either a prostaglandin F2{alpha} analog, or a GnRH antagonist, revealed that apoptosis of the luteal cells was associated with autophagocytosis. Likewise, degenerative changes that were not completely in accord with the morphologic features of apoptosis, but rather with autophagy, were observed during luteal regression in the cyclic human corpus luteum (Morales et al. 2000). Further studies need to be conducted to understand this nonapoptotic form of cell death in the regressive corpus luteum, as both forms of cell death (i.e., apoptosis and autophagocytosis) could occur concurrently. Whether the transformed luteal cells line used to demonstrate apoptosis in the present work could also be tested for the occurrence of autophagocytosis deserves a detailed investigation.

In conclusion, we have shown a parallelism between luteal cell death and the activation of a caspase-dependent apoptotic pathway, similar to what happens in vivo during luteal regression. Therefore, the in vitro model of immortalized rat luteal cells induced to die by serum starvation can be used to assess the relationship between apoptotic regulators and luteal regression and to study the overall mechanism of luteal cell death. Moreover, this defined experimental model of cell death could be used to study the fundamental mechanisms of apoptosis.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This research was supported by grant no. 2 P20 RR016479 from the INBRE Program, NIH/NCRR, and internal funds from the University of South Dakota School of Medicine. We are indebted to Dr Geula Gibori of the Department of Physiology and Biophysics, University of Illinois at Chicago, for kindly providing the GG-CL luteal cell line. We are also very grateful to Dr Barbara Goodman for the critical revision of the manuscript. Jacquelyn M Harmon was a student in the University of South Dakota Honors Program. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientifc work.


   Footnotes
 
Received 7 April 2005
First decision 30 June 2005
Revised manuscript received 5 July 2005
Accepted 5 August 2005


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

Abdo M, Hisheh S & Dharmarajan A 2003 Role of tumor necrosis factor-alpha and the modulating effect of the caspases in rat corpus luteum apoptosis. Biology of Reproduction 68 1241–1248.[Abstract/Free Full Text]

Al-Zi’abi MO, Fraser HM & Watson ED 2002 Cell death during natural and induced luteal regression in mares. Reproduction 123 67–77.[Abstract]

Bacci ML, Barazzoni AM, Forni M & Costerbosa GL 1996 In situ detection of apoptosis in regressing corpus luteum of pregnant sow: evidence of an early presence of DNA fragmentation. Domestic Animal Endocrinology 13 361–372.[CrossRef][ISI][Medline]

Bowen JM & Keyes PL 2000 Repeated exposure to prolactin is required to induce luteal regression in the hypophysectomized rat. Biology of Reproduction 63 1179–1184.[Abstract/Free Full Text]

Bowen JM, Keyes PL, Warren JS & Townson DH 1996 Prolactin-induced regression of the rat corpus luteum: expression of monocyte chemoattractant protein-1 and invasion of macrophages. Biology of Reproduction 54 1120–1127.[Abstract]

Bowen JM, Towns R, Warren JS & Keyes PL 1999 Luteal regression in the normally cycling rat: apoptosis, monocyte chemoattractant protein-1, and inflammatory cell involvement. Biology of Reproduction 60 740–746.[Abstract/Free Full Text]

Byskov AG 1974 Cell kinetic studies of follicular atresia in the mouse ovary. Journal of Reproduction and Fertility 37 277–285.[Medline]

Carambula SF, Matikainen T, Lynch MP, Flavell RA, Goncalves PB, Tilly JL & Rueda BR 2002 Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum. Endocrinology 143 1495–1501.[Abstract/Free Full Text]

Carambula SF, Pru JK, Lynch MP, Matikainen T, Goncalves PB, Flavell RA, Tilly JL & Rueda BR 2003 Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice. Reproductive Biology and Endocrinology 1 15.

Davis JS & Rueda BR 2002 The corpus luteum: an ovarian structure with maternal instincts and suicidal tendencies. Frontiers in Biosciences 7 1949–1978.[CrossRef]

Decker P & Muller S 2002 Modulating poly (ADP-ribose) polymerase activity: potential for the prevention and therapy of pathogenic situations involving DNA damage and oxidative stress. Current Pharmaceutical Biotechnology 3 275–283.[CrossRef][Medline]

Degli Esposti M, Ferry G, Masdehors P, Boutin JA, Hickman JA & Dive C 2003 Post-translational modification of Bid has differential effects on its susceptibility to cleavage by caspase 8 or caspase 3. Journal of Biological Chemistry 278 15749–15757.[Abstract/Free Full Text]

Dharmarajan AM, Hisheh S, Singh B, Parkinson S, Tilly KI & Tilly JL 1999 Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression. Endocrinology 140 2555–2561.[Abstract/Free Full Text]

Esposti MD 2002 The roles of Bid. Apoptosis 7 433–440.[CrossRef][ISI][Medline]

Fraser HM, Lunn SF, Harrison DJ & Kerr JB 1999 Luteal regression in the primate: different forms of cell death during natural and gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis. Biology of Reproduction 61 1468–1479.[Abstract/Free Full Text]

Gaytan F, Morales C, Bellido C, Aguilar E & Sanchez-Criado JE 1997 Role of prolactin in the regulation of macrophages and in the proliferative activity of vascular cells in newly formed and regressing rat corpora lutea. Biology of Reproduction 57 478–486.[Abstract]

Gaytan F, Bellido C, Morales C & Sanchez-Criado JE 1998 Both prolactin and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpus luteum of the rat. Biology of Reproduction 59 1200–1206.[Abstract/Free Full Text]

Gaytan F, Morales C, Bellido C, Aguilar R, Millan Y, Martin De Las Mulas J & Sanchez-Criado JE 2000 Progesterone on an oestrogen background enhances prolactin-induced apoptosis in regressing corpora lutea in the cyclic rat: possible involvement of luteal endothelial cell progesterone receptors. Journal of Endocrinology 165 715–724.[Abstract]

Gaytan F, Bellido C, Morales C & Sanchez-Criado JE 2001 Luteolytic effect of prolactin is dependent on the degree of differentiation of luteal cells in the rat. Biology of Reproduction 65 433–441.[Abstract/Free Full Text]

Goyeneche AA & Telleria CM 2005 Exogenous extradiol enhances apoptosis in regressing postpartum rat corpora lutea possibly mediated by prolactin. Reproductive Biology and Endocrinology 3 40.[CrossRef]

Goyeneche AA, Calvo V, Gibori G & Telleria CM 2002 Androstenedione interferes in luteal regression by inhibiting apoptosis and stimulating progesterone production. Biology of Reproduction 66 1540–1547.[Abstract/Free Full Text]

Goyeneche AA, Deis RP, Gibori G & Telleria CM 2003a Progesterone promotes survival of the rat corpus luteum in the absence of cognate receptors. Biology of Reproduction 68 151–158.[Abstract/Free Full Text]

Goyeneche AA, Martinez IL, Deis RP, Gibori G & Telleria CM 2003b In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro. Biology of Reproduction 68 2322–2330.[Abstract/Free Full Text]

Gulbins E, Dreschers S & Bock J 2003 Role of mitochondria in apoptosis. Experimental Physiology 88 85–90.[Abstract]

Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T & Alnemri ES 2002 Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. Journal of Biological Chemistry 277 13430–13437.[Abstract/Free Full Text]

Hehnke KE, Christenson LK, Ford SP & Taylor M 1994 Macrophage infiltration into the porcine corpus luteum during prostaglandin F2 alpha-induced luteolysis. Biology of Reproduction 50 10–15.[Abstract]

Hughes FM Jr & Gorospe WC 1991 Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 129 2415–2422.[Abstract]

Juengel JL, Garverick HA, Johnson AL, Youngquist RS & Smith MF 1993 Apoptosis during luteal regression in cattle. Endocrinology 132 249–254.[Abstract]

Khan SM, Dauffenbach LM & Yeh J 2000 Mitochondria and caspases in induced apoptosis in human luteinized granulosa cells. Biochemical and Biophysical Research Communications 269 542–545.[CrossRef][ISI][Medline]

Kuranaga E, Kanuka H, Bannai M, Suzuki M, Nishihara M & Takahashi M 1999 Fas/Fas ligand system in prolactin-induced apoptosis in rat corpus luteum: possible role of luteal immune cells. Biochemical and Biophysical Research Communications 260 167–173.[CrossRef][ISI][Medline]

Matsuyama S, Chang KT, Kanuka H, Ohnishi M, Ikeda A, Nishihara M & Takahashi M 1996 Occurrence of deoxyribonucleic acid fragmentation during prolactin-induced structural luteolysis in cycling rats. Biology of Reproduction 54 1245–1251.[Abstract]

McCormack JT, Friederichs MG, Limback SD & Greenwald GS 1998 Apoptosis during spontaneous luteolysis in the cyclic golden hamster: biochemical and morphological evidence. Biology of Reproduction 58 255–260.[Abstract/Free Full Text]

Morales C, Garcia-Pardo L, Reymundo C, Bellido C, Sanchez-Criado JE & Gaytan F 2000 Different patterns of structural luteolysis in the human corpus luteum of menstruation. Human Reproduction 15 2119–2128.[Abstract/Free Full Text]

Naftalin DM, Bove SE, Keyes PL & Townson DH 1997 Estrogen withdrawal induces macrophage invasion in the rabbit corpus luteum. Biology of Reproduction 56 1175–1180.[Abstract]

Papadopoulos V, Dharmarajan AM, Li H, Culty M, Lemay M & Sridaran R 1999 Mitochondrial peripheral-type benzodiazepine receptor expression. Correlation with gonadotropin-releasing hormone (GnRH) agonist-induced apoptosis in the corpus luteum. Biochemical Pharmacology 58 1389–1393.[CrossRef][ISI][Medline]

Rao KM, Meighan T & Bowman L 2002 Role of mitogen-activated protein kinase activation in the production of inflammatory mediators: differences between primary rat alveolar macrophages and macrophage cell lines. Journal of Toxicology and Environmental Health Part A 65 757–768.

Risk M & Gibori G 2001 Mechanisms of luteal cell regulation by prolactin. In Prolactin, pp 265–295. Ed. ND Horseman. New York: Kluwer Academic.

Rueda BR, Wegner JA, Marion SL, Wahlen DD & Hoyer PB 1995 Internucleosomal DNA fragmentation in ovine luteal tissue associated with luteolysis: in vivo and in vitro analyses. Biology of Reproduction 52 305–312.[Abstract]

Rueda BR, Tilly KI, Botros IW, Jolly PD, Hansen TR, Hoyer PB & Tilly JL 1997 Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression. Biology of Reproduction 56 186–193.[Abstract]

Shikone T, Yamoto M, Kokawa K, Yamashita K, Nishimori K & Nakano R 1996 Apoptosis of human corpora lutea during cyclic luteal regression and early pregnancy. Journal of Clinical Endocrinology and Metabolism 81 2376–2380.[Abstract]

Shintani T & Klionsky DJ 2004 Autophagy in health and disease: a double-edged sword. Science 306 990–995.[Abstract/Free Full Text]

Sugino N, Shimamura K, Tamura H, Ono M, Nakamura Y, Ogino K & Kato H 1996 Progesterone inhibits superoxide radical production by mononuclear phagocytes in pseudopregnant rats. Endocrinology 137 749–754.[Abstract]

Sugino N, Telleria CM & Gibori G 1997 Progesterone inhibits 20 alpha-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor. Endocrinology 138 4497–4500.[Abstract/Free Full Text]

Sugino N, Zilberstein M, Srivastava RK, Telleria CM, Nelson SE, Risk M, Chou JY & Gibori G 1998 Establishment and characterization of a simian virus 40-transformed temperature-sensitive rat luteal cell line. Endocrinology 139 1936–1942.[Abstract/Free Full Text]

Telleria CM, Zhong L, Deb S, Srivastava RK, Park KS, Sugino N, Park-Sarge OK & Gibori G 1998 Differential expression of the estrogen receptors alpha and beta in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 139 2432–2442.[Abstract/Free Full Text]

Telleria CM, Goyeneche AA, Cavicchia JC, Stati AO & Deis RP 2001 Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy. Endocrine 15 147–155.[CrossRef][ISI][Medline]

Tilly JL 1994 Ovarian follicular atresia: a model to study the mechanisms of physiological cell death. Endocrine 1 67–72.

Tilly JL, Kowalski KI, Johnson AL & Hsueh AJ 1991 Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 129 2799–2801.[Abstract]

Townson DH, Warren JS, Flory CM, Naftalin DM & Keyes PL 1996 Expression of monocyte chemoattractant protein-1 in the corpus luteum of the rat. Biology of Reproduction 54 513–520.[Abstract]

Van der Shepop HAM, van Diest PJ & Bagnell CA 1996 Counting of apoptotic cells: a methodological study in invasive breast cancer. Journal of Clinical Pathology and Molecular Pathology 49 M214–M217.

van Gurp M, Festjens N, van Loo G, Saelens X & Vandenabeele P 2003 Mitochondrial intermembrane proteins in cell death. Biochemical and Biophysical Research Communications 304 487–497.[CrossRef][ISI][Medline]

Yang H, Bhat GK & Sridaran R 2002 Clinostat rotation induces apoptosis in luteal cells of the pregnant rat. Biology of Reproduction 66 770–777.[Abstract/Free Full Text]

Yin XM 2000 Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Research 10 161–167.[CrossRef][ISI][Medline]

Young FM, Illingworth PJ, Lunn SF, Harrison DJ & Fraser HM 1997 Cell death during luteal regression in the marmoset monkey (Callithrix jacchus). Journal of Reproduction and Fertility 111 109–119.[Abstract]


This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. C. Peluffo, R. L. Stouffer, and M. Tesone
Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum
Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1215 - E1223.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
C. Stocco, C. Telleria, and G. Gibori
The Molecular Control of Corpus Luteum Formation, Function, and Regression
Endocr. Rev., February 1, 2007; 28(1): 117 - 149.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goyeneche, A. A
Right arrow Articles by Telleria, C. M
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goyeneche, A. A
Right arrow Articles by Telleria, C. M

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS