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Pregnancy |
a School of Anatomy and Human Biology, The University of Western Australia, Crawley, Western Australia 6009, Australia, and the West Australian Institute of Medical Research, Sir Charles Gairdner Hospital, Shenton Park, Western Australia 6008, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is a pleiotropic cytokine that has been implicated in apoptosis of many cell systems. However, the signal transduction of TNF during the structural and functional regression of the corpus luteum (CL) is largely unknown. In this study, we investigate the role of TNF in rat CL apoptosis and the involvement of monocyte chemoattractant protein-1 (MCP-1) and the modulating effect of the caspases in this process. An in vivo study of CL during pregnancy and postpartum using immunohistochemistry and Western blot analysis indicated that increases in TNF correspond with luteal apoptosis approaching term (Day 22) and at postpartum (Day 3). CL apoptosis was further investigated using a whole-CL culture model of tropic withdrawal. An increase was observed in both low molecular weight (MW) DNA fragmentation and TUNEL staining from 0 h to 8 h in culture. CL apoptosis in vitro was associated with increased protein expression of both TNF and MCP-1 as measured by immunohistochemistry and Western blot analysis. Using a whole-CL culture model, apoptosis was induced in vitro by TNF as demonstrated by a dose-dependent increase in DNA fragmentation. Treatment of luteal cells with TNF and both specific caspase inhibitors (Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK) or a general caspase inhibitor (Boc-D-FMK) prevented the effect of TNF. CL regression involves the apoptotic deletion of luteal cells; the results of this study suggest that TNF is possibly involved in this process. The observed increases in MCP-1 expression suggest the coordination of TNF expression with the infiltration and activation of macrophages. Furthermore, the results demonstrate the importance of the caspases in the TNF signal transduction pathway and suggest a hierarchy within the caspase family.
apoptosis, corpus luteum, cytokines, ovary, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine that is known to produce a signal that initiates apoptosis [12]. Locally produced cytokines such as TNF may induce follicular atresia [13] and CL apoptosis [14]. The expression of TNF within the ovary of the human [15], cow [16], mouse [17], and rat [13, 18] during the normal menstrual/estrous cycle is well documented. Cell types within the ovary that express TNF include macrophages [19, 20], granulosa cells [21, 22], and luteal cells [16, 23].
Macrophages are attracted to and infiltrate the CL during luteolysis [24], resulting in increased local production of cytokines. This accumulation of macrophages in the regressing CL is believed to be in response to a chemotactic factor; monocyte chemoattractant protein-1 (MCP-1) is a likely candidate for this chemotactic role. MCP-1 has been identified in luteal cells [25], and changes in its expression have been reported in the rat CL during estrus [26] and pregnancy [27]. In addition to the regulation of leukocyte migration and function, MCP-1 has a specific role in apoptosis [26] because it is regulated by local growth factors and cytokines such as TNF [28].
The binding of TNF to either of its receptors activates a signal transduction pathway of mammalian cysteine proteases that are central to the execution of the apoptotic cell death program [29, 30]. These enzymes are collectively referred to as caspases [31], and their proteolytic activity is characterized by their unusual ability to cleave proteins at aspartic acid residues. Active caspases can often activate other caspases, allowing initiation of a protease cascade [32]. To date, the caspase family consists of 14 homologues [33], each with varied substrates and cellular outcomes dependent on a specific tetrapeptide sequence [34].
The objective of this study was to determine the role of TNF during rat CL apoptosis, to investigate those factors such as MCP-1 that may influence TNF expression, and to elucidate the modulating role of the caspases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nulliparous albino Wistar rats, 3–5 mo old and weighing 261 ± 37 g (mean ± SD) at mating, were obtained from the Animal Resources Center (Murdoch, WA, Australia). Animals were housed under a 12L/12D schedule at 21°C and 55% humidity, with food and water provided ad libitum. Animals used in all protocols were mated overnight, with the following morning designated as Day 1, provided spermatozoa were present in a vaginal smear. Litters were born on Day 23 of pregnancy. All tissues were collected under aseptic conditions with light anesthesia using a mix of 0.2 L/min O2, 0.8 L/min NO, and 5% halothane. All procedures were reviewed and approved by the Animal Ethics Committee of The University of Western Australia.
Tropic Withdrawal
The in vitro organ culture system of tropic withdrawal is based on previous studies using rabbit CL [6, 35] and rat CL [36]. Three rats from Day 16 of pregnancy were used. Ovaries were excised and new CL dissected from the ovarian stroma. CL were cultured in pairs in serum-free minimal essential medium (MEM; Gibco BRL Life Technologies, Grand Island, NY) for 0 (snap frozen), 2, 4, 6, and 8 h at 37°C with 95% O2/5% CO2. A minimum of three CL pairs each from a different animal were used for each time point (n = 3). This procedure was repeated for use in immunohistochemical, Western blot, and DNA analyses.
TNF-Induced Apoptosis and Inhibition
Whole-CL culture
Three animals from Day 16 of pregnancy were used. CL were nonenzymatically excised from the ovary and cultured in pairs in MEM and 30% fetal bovine serum (FBS) supplemented with 1) medium alone (control) or 2) 12.5, 37.5, 75, or 125 ng/ml of recombinant rat TNF (R&D Systems, Minneapolis, MN). Following incubation for 6 h at 37°C with 95% O2/5% CO2, CL were processed as described under each experimental method (Western blot analysis and DNA 3'-end labeling). Three pairs each from a different animal were collected for each treatment group (n = 3).
Luteal cell culture
A primary cell culture was prepared from Day 16 CL as described previously [37] with modification. Briefly, CL were harvested from five animals (n = 1) and maintained at 37°C in dissection media (Hanks balanced salt solution; Gibco BRL) supplemented with BSA (2%) and HEPES (25 mM). CL were halved and incubated for 30 min at 37°C and 95% O2/5% CO2 in dissection media supplemented with collagenase IV, dispase, and DNase. The medium was replaced and CL were incubated for a further 30 min; this was repeated twice. Following this, the dissection media was replaced with 10 ml EDTA solution (PBS, 1% EDTA, 1% BSA, and 25 mM HEPES) and incubated at 37°C for 5 min, followed by centrifugation at 200 x g for 5 min. Luteal cells were resuspended in 10 ml of dissection medium and centrifuged at 200 x g for 5 min and repeated. The cell pellet was resuspended in 10 ml of dissection medium, passed through a silk screen, centrifuged at 200 x g for 5 min, and resuspended in 1 ml of Media 199 (Gibco BRL) supplemented with L-glutamine (2 mM), HEPES (25 mM), penicillin (1000 U/ml), streptomycin (1000 µg/ml), and FBS (5%). Cells were plated at approximately 700 000 cells/well, the medium was replaced following a 24-h incubation representing 0 h. Luteal cells were treated with 1) medium alone (control), 2) recombinant rat TNF (12.5, 25, or 37.5 ng/ml), and 3) recombinant rat TNF (37.5 ng/ml) with caspase inhibitor (50 µM).
Inhibitors used were specific inhibitors for caspases 1 (Z-YVAD-FMK), 2 (Z-VDVAD-FMK), 3 (Z-DEVD-FMK), 5 (Z-WEHD-FMK), 6 (Z-VEID-FMK), 8 (Z-IETD-FMK), 9 (Z-LEHD-FMK) and a general caspase inhibitor (Boc-D-FMK) (Calbiochem, Sydney, NSW, Australia). Following incubation for 6 h with the above treatments, cells were washed in PBS, centrifuged at 200 x g for 5 min, and snap frozen in liquid nitrogen. Following this, samples were processed as described for Western blot and DNA 3'-end labeling analyses. This was repeated three times (n = 3).
Immunohistochemistry
Localization of TNF within the CL was established using immunohistochemistry. Four rats from each stage of pregnancy (Days 16 and 22) and postpartum (Day 3) were used. One ovary (alternating left or right) from each animal was used for this study; the contralateral ovary was used for Western blot and DNA analyses. CL collected for immunocytochemistry were fixed in 4% paraformaldehyde at 4°C for 16 h, followed by postfixation in PBS (pH 7.4) at 4°C until processing. Tissue was paraffin embedded using a 10 h low-temperature (57°C) regime and 5-µm-thick sections were cut. Paraffin sections were dewaxed, washed twice in toluene, and rehydrated through a graded series of alcohol. Deparaffinized sections were rinsed in deionized water, treated with 2.5% periodic acid to inhibit endogenous peroxidases [38], washed in PBS, then incubated with 10% FBS for 1 h at room temperature. Sections were incubated with a 1:100 dilution of polyclonal rabbit anti-mouse TNF antibody (Genzyme Corporation, Cambridge, MA) for 2 h at 37°C. Sections were rinsed in PBS and incubated with a 1:200 dilution of sheep anti-rabbit IgG-HRP (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) for 1 h at 37°C and washed in PBS. All sections were then incubated in 3,3'-diaminobenzidine tetrahydrochloride (DAB; 1.2 mg/ml) for 10 min, counterstained in hematoxylin for 30 sec, and mounted. Negative controls were treated in the absence of the primary antibody. This procedure was repeated for each animal group (n = 4).
DNA 3'-End Labeling
Total cellular DNA was extracted from CL and luteal cells as originally described by Gross-Bellard et al. [39] and as modified by Roughton et al. [36]. DNA (1 µg) was labeled with [32P] ddATP (3000 Ci/mmol; Amersham Corporation, Arlington Heights, IL) using terminal transferase (Roche, Mannheim, Germany) as described previously [35] and separated on 2% agarose gel. Following electrophoresis, gels were dried in a slab gel drier without heat and exposed to autoradiography film (Kodak XAR-5; Eastman Kodak Company, Rochester, NY). Low molecular weight (MW) DNA fractions (<15 kb) were excised from the gel, mixed with 3 ml scintillation fluid, and counted in a ß counter to provide a quantitative estimate of the degree of internucleosomal DNA cleavage among samples. Three groups (n = 3) were assessed for each variable under investigation.
TUNEL Assay
CL collected following whole-CL organ culture for immunohistochemistry were prepared for TUNEL analysis as follows. Paraffin sections were dewaxed, washed twice in toluene, and rehydrated through a graded series of alcohol into deionized water. Sections were incubated with proteinase K (20 µg/ml) for 30 min at 37°C and rinsed in PBS. Using the In Situ Cell Death Detection Kit AP (Roche) sections were incubated with 50 µl TUNEL reaction mixture for 1 h at 37°C in a humidified chamber. Slides were washed in PBS and incubated with 50 µl converter AP at 37°C for 30 min. Slides were rinsed in PBS and incubated in 50–100 µl nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution under low light for 30 min at room temperature. Slides were rinsed in PBS, counterstained with eosin for 30 sec, and mounted. Negative control sections were incubated with 50 µl of the TUNEL reaction mixture without terminal transferase. Sections used as positive controls were incubated at 37°C for 10 min with DNase (1 µg/ml) following treatment with proteinase K. Three CL pairs were assessed for each experimental time point (n = 3).
Western Blot Analyses
Tissues were homogenized in sodium phosphate buffer (10 mM, pH 7) containing sucrose (0.25 M), EDTA (1 mM), PMSF (1 mM), and trypsin inhibitor (100 mg/ml). Protein concentration of homogenates was measured [40] and 30 µg resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes (MSI, Westboro, MA). Membranes were blocked in 5% nonfat milk in Tris-buffered saline/0.1% Tween-20 (TBST) for 1 h at room temperature. Membranes were then separately probed with antibodies against TNF (1:2000; Genzyme Corporation) for 1–2 h at room temperature and MCP-1 (1:500, biotinylated mouse anti-rat MCP-1 monoclonal antibody; BD PharMingen, Franklin Lakes, NJ) in TBST overnight at 4°C. Following primary incubation, membranes were washed in TBST and incubated with a secondary antibody (1:40 000 goat anti-rabbit IgG [Santa Cruz Biotechnology, Santa Cruz, CA] and/or streptavidin horseradish peroxidase) for 1 h at room temperature. Protein signals were detected by enhanced chemiluminescence (Supersignal West Pico ECL substrate; Pierce, Rockford, IL) and exposed to autoradiographic film (Kodak XAR-5). Control procedures included the omission of the primary antibody during incubation. A common tissue sample was included on each gel to allow for standardization of chemiluminescence levels and exposure times. Following densitometric measurement, the efficiency of protein transfer was assessed by staining of each gel (posttransfer) with Coomassie Brilliant Blue (Sigma) and sample loading assessed (qualitatively) by staining of membranes following incubation. This procedure was repeated for each animal/experimental group (n = 3) for both TNF and MCP-1 analyses.
Statistical Analyses
All experiments were conducted using a minimum of three animals per time point/treatment. Quantitative results were obtained from counts per million (CPM) of radiolabeled low-MW DNA fragments (DNA 3'-end labeling) and densitometric counts of autoradiograms (Western blot analysis). Statistical analyses are presented in graphical form, and where appropriate, an autoradiogram, which best represents the result of several repeats, is presented. Results are expressed as means ± SEM (fold changes against control group). Variation among groups was analyzed by one-way ANOVA. When significant differences (P < 0.05) among groups were detected, specific group comparisons were made by least significant difference (LSD) tests. Associations between parameters were measured by Pearson correlation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was evident in CL on Days 16 and 22 of pregnancy and Day 3 postpartum (Fig. 1, a, b, and c, respectively). Immunostaining within the CL compartment was confined to the cytoplasm of luteal cells (Fig. lc). Qualitative assessment of the results indicated that TNF expression increased toward parturition and postpartum. The most intense sites of immunoreactivity observed within the adult rat ovary during pregnancy and postpartum were confined to the ovarian stroma. In particular, staining of macrophage-like cells was evident (Fig. 1d). Control sections treated in the absence of the primary antibody showed no nonspecific staining (Fig. 1e). The staining pattern and intensity were consistent between immunohistochemical runs.
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Western blot analysis using a polyclonal rabbit anti-mouse TNF antibody revealed a single immunoreactive band of approximately 17 kDa consistent with the cleaved TNF protein (Fig. 2a). This signal was evident on Days 16 and 22 of pregnancy and Day 3 postpartum. Statistical analysis of arbitrary densitometric counts (Fig. 2b) indicated a significant increase in the level of TNF protein on Day 22 of pregnancy and Day 3 postpartum relative to Day 16 (P < 0.05).
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DNA 3'-end labeling, TUNEL assay, and TNF immunohistochemistry of CL following incubation without tropic support are shown in Figure 3 (a, b, and c, respectively). Apoptotic DNA fragmentation was evident as a DNA ladder following 8 h of incubation (Fig. 3a). Statistical analysis of CPM of radiolabeled low-MW DNA fragments for all experimental time points demonstrated a significant increase over the time course of 8 h relative to 0 h (mean fold change against control (0 h) = 14.91, P < 0.05). DNA fragmentation was also demonstrated in situ through TUNEL assay (Fig. 3b). DNA fragmentation within dying cells was observed as dark staining nuclei (arrow). Snap frozen CL (0 h) showed no DNA fragmentation. In comparison, CL incubated for 8 h demonstrated numerous dark-staining nuclei throughout the CL compartment (2-, 4-, and 6-h time points not shown). Immunohistochemical staining indicated that TNF was present in luteal cells (lc) undergoing spontaneous apoptosis (Fig. 3c). An increase (qualitative) in the level of staining intensity was observed from 0 to h (2-, 4-, and 6-h time points not shown). Negative controls, treated in the absence of the primary antibody, showed no nonspecific staining (not shown). The staining pattern and intensity were consistent between immunohistochemical runs.
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Western blot analysis of TNF and MCP-1 protein expression revealed a clear immunoreactive signal at the expected size for both TNF (17 kDa) and MCP-1 (12 kDa) (Fig. 4a). A significant increase in TNF signal was observed from 0 to 8 h (Fig. 4b). A positive association (r = 0.86; P < 0.05) was observed between TNF expression and the degree of DNA fragmentation observed following tropic withdrawal. An increase in MCP-1 expression was observed following 8 h of incubation without tropic support compared with 0 h, though this failed to reach significance. MCP-1 expression correlated (r = 0.89) with an increase in apoptotic DNA fragmentation following tropic withdrawal, though this was not significant (P < 0.07).
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TNF treatment in vitro indicated a dose-dependent increase in the level of DNA fragmentation (Fig. 5a). CPM of radiolabeled low-MW DNA fragments demonstrated a significant increase in the level of apoptotic DNA fragmentation at 37.5 and 75 ng/ml relative to control (mean fold change against control 6 h = 1.61 and 2.31, respectively; P < 0.05). Corresponding MCP-1 protein levels were investigated through Western blot analysis (Fig. 5b). MCP-1 protein expression correlated (r = 0.84) with TNF-induced apoptosis. The strongest immunoreactive band corresponded with the greatest fold change following TNF treatment (75 ng/ml), although across all treatments, this was not significant (P < 0.07). A weaker immunoreactive signal was apparent at approximately 16 kDa, though this was attributed to the nonspecific staining of the secondary antibody on this occasion.
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Luteal cells were incubated for 6 h in the absence (control) or presence of TNF (12.5, 25, and 37.5 ng/ml) and TNF (37.5 ng/ml) and a specific caspase inhibitor (Fig. 6). Levels of apoptotic DNA fragmentation were assessed by CPM of radiolabeled low-MW DNA fragments. Apoptotic DNA fragmentation increased (mean fold change against control 6 h = 6.44) following treatment with recombinant TNF (37.5 ng/ml) (12.5 and 25 ng/ml treatments not shown) (Fig. 6a). A significant difference (P < 0.05) in DNA fragmentation was observed between luteal cells treated with TNF alone and those treated with TNF and inhibitors of caspases 3 (Z-DEVD-FMK), 6 (Z-VEID-FMK), 8 (Z-IETD-FMK) and a general caspase inhibitor (Boc-D-FMK) (Fig. 6b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The expression of TNF by luteal cells was anticipated as a consequence of their origin. Previous studies have established granulosa cells as both a putative source and biological target for TNF [22, 41]. Furthermore, the presence of TNF during the estrous cycle in several species is well documented [16, 42]. Our immunohistochemical and Western blot analyses demonstrated TNF expression during both functional and induced apoptosis in the CL of pregnancy and postpartum. Macrophage-like cells within the ovarian stroma were also stained positive.
Macrophages are the primary source of TNF throughout the body and have been localized within the ovary [21] and the CL compartment [43, 44]. Several studies have reported that macrophage numbers within the CL increase corresponding to the age of the CL and the progression of luteolysis [19, 45]. The trigger for the infiltration of macrophages is unknown, though MCP-1 has been demonstrated as a potential candidate [46]. MCP-1 expression was shown to correlate with increasing CL apoptosis in a time-dependent manner. Moreover, our data demonstrated MCP-1 expression corresponds with increasing TNF-induced apoptosis in a dose-dependent manner, though this was not significant. However, it is plausible that MCP-1 and the infiltration of macrophages into the CL influence TNF expression and subsequently CL apoptosis and that this interaction may occur via positive feedback [28, 47]. The primary function of macrophages in the CL is the phagocytosis of degenerative luteal cells; however, they are also responsible for the local production of cytokines. TNF expression may increase both MCP-1 expression and macrophage numbers, subsequently accelerating the rate of apoptosis.
The treatment of both luteal cells and CL with recombinant TNF (37.5 and 75 ng/ml, respectively) was able to induce apoptosis in vitro. Varied concentrations of TNF up to 300 ng/ml have been reported [13, 48–51], although high concentrations appear to have a greater influence on the timing of the apoptotic event rather than the degree of DNA fragmentation.
We have previously demonstrated the role of the caspases during spontaneous CL apoptosis following tropic withdrawal in vitro in the rabbit [35]. Treatment with sodium aurothiomalate (SAM) prevented the spontaneous onset of apoptotic DNA fragmentation. However, the mechanisms of inhibition are unknown and are probably compound specific [52]. SAM is regarded as a specific, noncompetitive inhibitor of caspase 1 and related proteases [51, 53] and is therefore unable to distinguish the individual roles of caspases 1, 4, 5, 7, and 9.
A caspase hierarchy has been proposed that divides caspases into either initiator or effector caspases [54]. Using the specificity of the caspases, we were able to identify the relative importance of each during TNF-induced CL apoptosis using specific caspase inhibitors. Specific caspase inhibitors act by binding to the active site of the caspases because their design includes a peptide recognition sequence and a functional group. The peptide recognition sequence corresponds to those found on endogenous substrates. Incubation of luteal cells in the presence of inhibitors of caspases 3 (Z-DEVD-FMK), 6 (Z-VEID-FMK), 8 (Z-IETD-FMK) and a general caspase inhibitor (Boc-D-FMK) resulted in a reduction in the level of TNF-induced DNA fragmentation. Caspase 3 has been shown to play a critical role during development and apoptosis [32]. Caspase 6, a member of the CED-3 subfamily of caspases with a high homology to caspase 3, has been implicated in apoptosis [55, 56], and caspase 8 was identified as a protein recruited following activation of cytokine cell surface receptors. Recombinant caspase 8 is able to process all known caspases [57, 58], thereby serving as a link to the proapoptotic proteases of the caspase family.
The inability of certain caspase inhibitors to significantly inhibit TNF-induced apoptosis was unexpected. As an effector caspase, caspase 3 is believed responsible for the cleavage of structural proteins within the cell and ultimately the apoptotic phenotype [59, 60]. Thus, caspase 3 inhibition was expected to correlate with the greatest decrease in DNA fragmentation [61]. However, general inhibition of the caspases was more effective than specific inhibition in reducing the level of DNA fragmentation, supporting the proteolytic cascade of caspase signal transduction. The differing efficacy of specific caspase inhibition suggests the placement of the caspases within the cascade. Inhibition of caspases 6 and 8, which are reportedly apical caspases in the cascade, demonstrated the greatest reduction in DNA fragmentation. Further investigation of the cleaved protein levels of each caspase under the same experimental conditions, including the inhibition of initiator caspases, will conclusively determine this hierarchy.
The findings of this study do not allow us to speculate about which ovarian factor drives the process of CL apoptosis. While the expression of MCP-1 and the infiltration of macrophages appears important, in order to fully explain the event of luteolysis, other factors must be clarified. The experimental components of this study do suggest that luteal cells are capable of endogenous TNF expression. The localization of TNF protein in the CL suggests that luteal cells and other ovarian sources are capable of TNF synthesis, while the ability of recombinant TNF to induce apoptosis suggests that CL apoptosis is enacted in part through the TNF cell-death pathway. Observed increases in MCP-1 expression suggest a coordinated induction of TNF expression through the infiltration and activation of macrophages. Furthermore, the results demonstrate the importance of the caspases within the TNF signal transduction pathway and the importance of both initiator and/or effector caspases in CL apoptosis.
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FOOTNOTES |
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2 Correspondence: A.M. Dharmarajan, School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. FAX: 61 0 8 9380 1051; dharma{at}anhb.uwa.edu.au
Received: 29 August 2002.
First decision: 17 September 2002.
Accepted: 18 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) production and localization of macrophages and T lymphocytes in the rabbit corpus luteum. Endocrinology 1988 122:1185-1187This article has been cited by other articles:
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  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]
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 C. R. Greenfeld, K. F. Roby, M. E. Pepling, J. K. Babus, P. F. Terranova, and J. A. Flaws Tumor Necrosis Factor (TNF) Receptor Type 2 Is an Important Mediator of TNF alpha Function in the Mouse Ovary Biol Reprod, February 1, 2007; 76(2): 224 - 231. [Abstract] [Full Text] [PDF]
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 C. R Greenfeld, J. K Babus, P. A Furth, S. Marion, P. B Hoyer, and J. A Flaws BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries Reproduction, January 1, 2007; 133(1): 107 - 116. [Abstract] [Full Text] [PDF]
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M. J. Cannon and J. L. Pate Indoleamine 2,3-Dioxygenase Participates in the Interferon-gamma-Induced Cell Death Process in Cultured Bovine Luteal Cells Biol Reprod, March 1, 2006; 74(3): 552 - 559. [Abstract] [Full Text] [PDF]
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A. A Goyeneche, J. M Harmon, and C. M Telleria Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis Reproduction, January 1, 2006; 131(1): 103 - 111. [Abstract] [Full Text] [PDF]
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S. Takiguchi, N. Sugino, K. Esato, A. Karube-Harada, A. Sakata, Y. Nakamura, H. Ishikawa, and H. Kato Differential Regulation of Apoptosis in the Corpus Luteum of Pregnancy and Newly Formed Corpus Luteum after Parturition in Rats Biol Reprod, February 1, 2004; 70(2): 313 - 318. [Abstract] [Full Text] [PDF]
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